indel mutation patterns dna  (New England Biolabs)


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    New England Biolabs indel mutation patterns dna
    Forcing CtIP recruitment to the <t>DNA</t> cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and <t>indel</t> frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P
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    1) Product Images from "CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair"

    Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03475-7

    Forcing CtIP recruitment to the DNA cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and indel frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P
    Figure Legend Snippet: Forcing CtIP recruitment to the DNA cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and indel frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P

    Techniques Used: Sequencing, Transfection, FACS, Cotransfection

    2) Product Images from "Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2"

    Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2018.05.018

    SETD1A Suppresses Fork Degradation after Replication Stress (A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post transfection, cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). DNA was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1 .
    Figure Legend Snippet: SETD1A Suppresses Fork Degradation after Replication Stress (A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post transfection, cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). DNA was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1 .

    Techniques Used: Transfection

    3) Product Images from "Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride"

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-36396-y

    Identification of the telomeric DNA sequence in A. viride . Dot blotting of A. viride genomic DNA (gDNA) using oligonucleotide ( a ) or double-stranded ( b ) telomeric probes. Control oligonucleotides and genomic DNA were included for comparison. The oligo dT 48 and bacterial genomic DNA are negative controls. ( c ) Telomeric sequences of A. viride are located at the ends of genomic DNA. Genomic DNA of A. viride was treated with Bal-31 exonuclease and then subjected to TRF assay using a double-stranded TTAGGG telomeric probe. In: intact genomic DNA. I: internal repetitive sequences. ( d,e ) A. viride telomeres detected by FISH in interphase nuclei ( d ) and metaphase spreads ( e ). Red fluorescent signals denote the TTAGGG telomeric sequences. Nuclei and chromosomes are in blue (DAPI). Scale bars: 10 μm.
    Figure Legend Snippet: Identification of the telomeric DNA sequence in A. viride . Dot blotting of A. viride genomic DNA (gDNA) using oligonucleotide ( a ) or double-stranded ( b ) telomeric probes. Control oligonucleotides and genomic DNA were included for comparison. The oligo dT 48 and bacterial genomic DNA are negative controls. ( c ) Telomeric sequences of A. viride are located at the ends of genomic DNA. Genomic DNA of A. viride was treated with Bal-31 exonuclease and then subjected to TRF assay using a double-stranded TTAGGG telomeric probe. In: intact genomic DNA. I: internal repetitive sequences. ( d,e ) A. viride telomeres detected by FISH in interphase nuclei ( d ) and metaphase spreads ( e ). Red fluorescent signals denote the TTAGGG telomeric sequences. Nuclei and chromosomes are in blue (DAPI). Scale bars: 10 μm.

    Techniques Used: Sequencing, TRF Assay, Fluorescence In Situ Hybridization

    4) Product Images from "RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation"

    Article Title: RG108 increases NANOG and OCT4 in bone marrow-derived mesenchymal cells through global changes in DNA modifications and epigenetic activation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0207873

    Changes in DNA modifications at gene-specific regulatory elements in response to RG108 treatment. Methylation and hydroxymethylation levels were analyzed by DNA glycosylation followed by restriction enzyme analysis and qPCR of promoter sequences, after 3 days of 50 μM RG108 treatment and compared to DMEM and DMSO controls. The relative levels were determined using the cycle threshold (Ct) method and the methylation results are presented as HpaII levels— MspI levels/control levels and the hydroxymethylation results are presented as MspI levels/control levels (% of control). Two biological replicates were performed for each group and each of the biological replicates was done in technical triplicates. (A) All DNMT genes are mostly unmethylated (20–30% methylation level) and undergo only small (2–3%) methylation after RG108. (B) The hydroxymethylation levels at DNMTs’ promoters are low and do not change after RG108 treatment. (C) TET genes have similar 20–30% methylation levels; TET1 and TET2 undergo small but significant demethylation after RG108. (D) Hydroxymethylation levels at TET genes are low and do not change with RG108. (E) RG108 treatment resulted in 40% and 40% loss of methylation and hydroxymethylation, respectively at NANOG regulatory element. (F) For OCT4, the methylation loss was 48% and hydroxymethylation was 32%. For all graphics, *, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. *, ** and *** indicate, respectively, p ≤ 0.01, p ≤ 0.001 p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.
    Figure Legend Snippet: Changes in DNA modifications at gene-specific regulatory elements in response to RG108 treatment. Methylation and hydroxymethylation levels were analyzed by DNA glycosylation followed by restriction enzyme analysis and qPCR of promoter sequences, after 3 days of 50 μM RG108 treatment and compared to DMEM and DMSO controls. The relative levels were determined using the cycle threshold (Ct) method and the methylation results are presented as HpaII levels— MspI levels/control levels and the hydroxymethylation results are presented as MspI levels/control levels (% of control). Two biological replicates were performed for each group and each of the biological replicates was done in technical triplicates. (A) All DNMT genes are mostly unmethylated (20–30% methylation level) and undergo only small (2–3%) methylation after RG108. (B) The hydroxymethylation levels at DNMTs’ promoters are low and do not change after RG108 treatment. (C) TET genes have similar 20–30% methylation levels; TET1 and TET2 undergo small but significant demethylation after RG108. (D) Hydroxymethylation levels at TET genes are low and do not change with RG108. (E) RG108 treatment resulted in 40% and 40% loss of methylation and hydroxymethylation, respectively at NANOG regulatory element. (F) For OCT4, the methylation loss was 48% and hydroxymethylation was 32%. For all graphics, *, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. *, ** and *** indicate, respectively, p ≤ 0.01, p ≤ 0.001 p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction

    Global effects of RG108 on DNA modifications and DNMTs and TETs enzymatic activities in hBMSCs. hBMSCs were treated with 50 μM RG108 for 3 days and compared to DMEM and DMSO controls. (A) Global methylation was assessed using Imprint Methylated DNA Quantification Kit showing a significant decrease in the RG108 group. (B) Global hydroxymethylation was analyzed using Quest 5-hmC DNA Elisa kit; no statistical differences are observed amongst groups. DNMTs (C) and TETs (D) enzymatic activities were assessed using colorimetric assays. Global methylation and hydroxymethylation experiments were performed in biological triplicates and DNMTs and TETs activities were performed in biological duplicates. For all graphics, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. ** and *** indicate, respectively, p ≤ 0.001 and p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.
    Figure Legend Snippet: Global effects of RG108 on DNA modifications and DNMTs and TETs enzymatic activities in hBMSCs. hBMSCs were treated with 50 μM RG108 for 3 days and compared to DMEM and DMSO controls. (A) Global methylation was assessed using Imprint Methylated DNA Quantification Kit showing a significant decrease in the RG108 group. (B) Global hydroxymethylation was analyzed using Quest 5-hmC DNA Elisa kit; no statistical differences are observed amongst groups. DNMTs (C) and TETs (D) enzymatic activities were assessed using colorimetric assays. Global methylation and hydroxymethylation experiments were performed in biological triplicates and DNMTs and TETs activities were performed in biological duplicates. For all graphics, ** or *** above the bars represent significant inter-group differences when compared to DMEM. Other significant differences are represented by * symbol above the linkers. ** and *** indicate, respectively, p ≤ 0.001 and p ≤ 0.0001 by ANOVA One Way followed by the Tukey test.

    Techniques Used: Methylation, Enzyme-linked Immunosorbent Assay

    5) Product Images from "Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing"

    Article Title: Fragmentation Through Polymerization (FTP): A new method to fragment DNA for next-generation sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0210374

    Agarose-gel electrophoresis of gDNA fragmented by the FTP method. gDNA of E . coli BL21 was incubated as described in Materials and Methods: without enzymes (lane 1), with SD polymerase (lane 2), with DNase I (lane 3), and with both DNase I and SD polymerase (lane 4 and 5). M1: 1 kb DNA Ladder; M2: 100 bp DNA Ladder.
    Figure Legend Snippet: Agarose-gel electrophoresis of gDNA fragmented by the FTP method. gDNA of E . coli BL21 was incubated as described in Materials and Methods: without enzymes (lane 1), with SD polymerase (lane 2), with DNase I (lane 3), and with both DNase I and SD polymerase (lane 4 and 5). M1: 1 kb DNA Ladder; M2: 100 bp DNA Ladder.

    Techniques Used: Agarose Gel Electrophoresis, Incubation

    A general overview of the dsDNA Fragmentation Through Polymerization (FTP) method. The FTP method is based on two enzymatic reactions: a DNA nicking reaction with DNase I and a strand-displacement DNA polymerization with SD DNA polymerase. As a result, multiple double-stranded DNA fragments with overlapping sequences are generated. De novo synthesized DNA is indicated in grey, and SD polymerase is indicated in red.
    Figure Legend Snippet: A general overview of the dsDNA Fragmentation Through Polymerization (FTP) method. The FTP method is based on two enzymatic reactions: a DNA nicking reaction with DNase I and a strand-displacement DNA polymerization with SD DNA polymerase. As a result, multiple double-stranded DNA fragments with overlapping sequences are generated. De novo synthesized DNA is indicated in grey, and SD polymerase is indicated in red.

    Techniques Used: Generated, Synthesized

    6) Product Images from "The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication"

    Article Title: The Dictyostelium discoideum homologue of Twinkle, Twm1, is a mitochondrial DNA helicase, an active primase and promotes mitochondrial DNA replication

    Journal: BMC Molecular Biology

    doi: 10.1186/s12867-018-0114-7

    Helicase activity and substrate preference of D. discoideum Twm1. In vitro helicase activity of Twm1 was determined at 21 °C using various fluorescently labelled dsDNA templates (Additional file 3 : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)
    Figure Legend Snippet: Helicase activity and substrate preference of D. discoideum Twm1. In vitro helicase activity of Twm1 was determined at 21 °C using various fluorescently labelled dsDNA templates (Additional file 3 : Table S1B). Each DNA template was heated to 100 °C (H; first lane) and assayed using a no protein negative control (N; empty vector purification; second lane) in addition to Twm1 (T; third lane). Substrate (S) and final product (P) are indicated. Overhang polarities and FAM labels (red dots) of substrates are also indicated. a Helicase assay using strict dsDNA (FHA0) or open fork-like dsDNA (5′ and 3′ overhangs; FHAOF). b Determination of Twm1 directionality using open fork-like dsDNA with one duplex overhang (FHAOF5 or FHAOF3). c Overhang requirements of Twm1 were determined using dsDNA with a single ssDNA overhang (5′ or 3′; FHA5 or FHA3, respectively). Directionality of Twm1 was reconfirmed by using a duplex 3′ overhang (FHA3D)

    Techniques Used: Activity Assay, In Vitro, Negative Control, Plasmid Preparation, Purification, Helicase Assay

    7) Product Images from "The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma"

    Article Title: The degree of mitochondrial DNA methylation in tumor models of glioblastoma and osteosarcoma

    Journal: Clinical Epigenetics

    doi: 10.1186/s13148-018-0590-0

    mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of
    Figure Legend Snippet: mtDNA methylation identified by pyro-sequencing. Levels of DNA methylation at CpG sites in a HSP, b LSP, and c ND6 regions of 143B cells, 143B 143B early and 143B 143B late tumors were determined by pyro-sequencing. Statistical significance was determined by One-way ANOVA. Bars represent the mean of the percentage of DNA methylation (mean ± SEM; n = 3). * and ** indicate p values of

    Techniques Used: Methylation, Sequencing, DNA Methylation Assay

    8) Product Images from "Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis"

    Article Title: Quantitative disclosure of DNA knot chirality by high-resolution 2D-gel electrophoresis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz015

    Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.
    Figure Legend Snippet: Test of the electrophoresis procedure that discerns DNA knot chirality. ( A ) A linear 4.4-kb DNA fragment was circularized in free solution with T4 DNA ligase to produce a small fraction of molecules containing a trefoil knot. Negative supercoils were subsequently introduced by incubating the circularized DNA with topoisomerase I in presence of 250 μg/ml chloroquine. ( B ) The gel-blot shows the DNA products after high resolution 2D-gel electrophoresis carried out in 0.9% agarose gel (40 × 23 cm) in TBE. The first gel dimension (top to bottom) was run at 80 V for 70 h in TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA). The second gel dimension (left to right) was run at 120 V for 10 h in TBE containing 0.65 μg/ml of chloroquine. Lk, linking number topoisomers. N, nicked unknotted circles. L, linear DNA. The enlarged gel section shows the signal of Lk topoisomers of unknotted molecules (Kn# 0) and of molecules containing either a positive- or negative-noded trefoil knot (Kn# 3). ( C ) Probability of the two chiral forms of the trefoil knot.

    Techniques Used: Electrophoresis, Western Blot, Two-Dimensional Gel Electrophoresis, Agarose Gel Electrophoresis

    9) Product Images from "High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division"

    Article Title: High-throughput sequencing of sorted expression libraries reveals inhibitors of bacterial cell division

    Journal: BMC Genomics

    doi: 10.1186/s12864-018-5187-7

    High-throughput DNA sequencing of plasmid libraries from the reference-unsorted and filamentous-sorted populations. a Comparison of the distribution of mapped reads in 10 kb UTI89 chromosomal bins, showing the degree of enrichment of mapped reads in specific bins after flow cytometry. The dotted line indicates 20% of the mapped reads (see text). b The distribution of read depth along the ~ 5.06 Mbp UTI89 chromosome in the filamentous-sorted sample (red) and the reference-unsorted sample (blue), with a bin size of 1 kb. c The proportion of enriched regions found in common in both biological replicates versus their corresponding threshold significance score (−log 10 [ P -value]) (left-hand y-axis), and the total number of enriched regions from both replicates (right-hand y -axis). The score of 70 (dotted line) represents the selected threshold criteria for display in Table 2 . d The number of reads mapped to the region 1,295,150 – 1,302,200 bp of UTI89 in the filamentous-sort (red) and the reference-unsort (blue) samples within a bin window of 50 bp. Large read depth numbers in the filamentous-sort sample (replicate 1, red) can be seen against the reference-unsorted sample in the region spanning from the ycgI homolog to the minCDE genes (the region identified by MACS is shown by the bracket). Open reading frames (grey boxes) encoded by the upper and lower strands are indicated by their relative positioning below the axis
    Figure Legend Snippet: High-throughput DNA sequencing of plasmid libraries from the reference-unsorted and filamentous-sorted populations. a Comparison of the distribution of mapped reads in 10 kb UTI89 chromosomal bins, showing the degree of enrichment of mapped reads in specific bins after flow cytometry. The dotted line indicates 20% of the mapped reads (see text). b The distribution of read depth along the ~ 5.06 Mbp UTI89 chromosome in the filamentous-sorted sample (red) and the reference-unsorted sample (blue), with a bin size of 1 kb. c The proportion of enriched regions found in common in both biological replicates versus their corresponding threshold significance score (−log 10 [ P -value]) (left-hand y-axis), and the total number of enriched regions from both replicates (right-hand y -axis). The score of 70 (dotted line) represents the selected threshold criteria for display in Table 2 . d The number of reads mapped to the region 1,295,150 – 1,302,200 bp of UTI89 in the filamentous-sort (red) and the reference-unsort (blue) samples within a bin window of 50 bp. Large read depth numbers in the filamentous-sort sample (replicate 1, red) can be seen against the reference-unsorted sample in the region spanning from the ycgI homolog to the minCDE genes (the region identified by MACS is shown by the bracket). Open reading frames (grey boxes) encoded by the upper and lower strands are indicated by their relative positioning below the axis

    Techniques Used: High Throughput Screening Assay, DNA Sequencing, Plasmid Preparation, Flow Cytometry, Cytometry, Magnetic Cell Separation

    Overexpression of the pptE and pdhR ORFs cause E. coli filamentation independent of recA (SOS response). The pptE and pdhR ORFs from UTI89 were cloned into pBAD24, transformed into BW25113, and BW25113 (Δ recA ), and cultures induced with 0.2% L-arabinose in M9 medium. Fixed cells were analysed by microscopy ( a ) and Coulter cytometry to obtain cell volume distributions ( b ). c Fixed cells were stained using the Hoechst 33342 (DNA) and FM4–64 (membrane) and imaged by fluorescence microscopy. An overlay of the Hoechst and FM4–64 channels is also shown, indicating the obvious anti-correlation between the two stains, suggesting a possible physical exclusion of these features
    Figure Legend Snippet: Overexpression of the pptE and pdhR ORFs cause E. coli filamentation independent of recA (SOS response). The pptE and pdhR ORFs from UTI89 were cloned into pBAD24, transformed into BW25113, and BW25113 (Δ recA ), and cultures induced with 0.2% L-arabinose in M9 medium. Fixed cells were analysed by microscopy ( a ) and Coulter cytometry to obtain cell volume distributions ( b ). c Fixed cells were stained using the Hoechst 33342 (DNA) and FM4–64 (membrane) and imaged by fluorescence microscopy. An overlay of the Hoechst and FM4–64 channels is also shown, indicating the obvious anti-correlation between the two stains, suggesting a possible physical exclusion of these features

    Techniques Used: Over Expression, Clone Assay, Transformation Assay, Microscopy, Cytometry, Staining, Fluorescence

    Analysis and purification of filamentous bacteria from a UPEC DNA-expression library by flow cytometry. Flow cytometry was carried out with the control populations of strains JW0941–1/pBAD24 ( a ) and JW0941–1/pLau80 (containing FtsZ-YFP, which induces strong filamentation) ( b ). The sorted populations of the UTI89 gDNA expression library in JW0941–1 are also shown: an initial high-speed, high-yield sort ( c ) and a subsequent lower speed “purity sort” to further enrich for filamentous cells ( d ). Dots represent single events plotted as side-scatter values of peak height and peak width, which correlates with filament length. Event density is colour coded with a heat-map of red (high density) to blue (displaying individual events). The gates for sorting are indicated with vertical lines, and the percentages of events contained within that gate are indicated. The “short” gate was determined to encompass greater than 99% of the control bacterial population (short cells), JW0941–1/pBAD24 ( a )
    Figure Legend Snippet: Analysis and purification of filamentous bacteria from a UPEC DNA-expression library by flow cytometry. Flow cytometry was carried out with the control populations of strains JW0941–1/pBAD24 ( a ) and JW0941–1/pLau80 (containing FtsZ-YFP, which induces strong filamentation) ( b ). The sorted populations of the UTI89 gDNA expression library in JW0941–1 are also shown: an initial high-speed, high-yield sort ( c ) and a subsequent lower speed “purity sort” to further enrich for filamentous cells ( d ). Dots represent single events plotted as side-scatter values of peak height and peak width, which correlates with filament length. Event density is colour coded with a heat-map of red (high density) to blue (displaying individual events). The gates for sorting are indicated with vertical lines, and the percentages of events contained within that gate are indicated. The “short” gate was determined to encompass greater than 99% of the control bacterial population (short cells), JW0941–1/pBAD24 ( a )

    Techniques Used: Purification, Expressing, Flow Cytometry, Cytometry

    10) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.
    Figure Legend Snippet: Genome-wide base composition bias curves in Illumina reads from PCR-free human DNA libraries. ( a ) The GC-bias curves from libraries (in duplicate) produced by the immobilized enzyme method (IM-1 and IM-2 in blue), for end repair for 30 min at 20 °C and 3′ A-tailing at 37 °C in contrast to the data from the libraries generated by the soluble enzyme method, with 3′ A-tailing at 65 °C, using enzyme mixture PKT (PKT-1 and PKT-2 in purple). ( b ) The GC-bias data of the immobilized enzyme method compared to the data from the duplicate libraries generated by Illumina TruSeq DNA PCR-free LT Library Preparation Kit (Illumina), Kapa Hyper Prep Kit (Kapa) or NEBNext Ultra II DNA Library Prep Kit for Illumina (Ultra) according to the protocols of the manufacturers. The Illumina protocol carries out end repair for 30 min at 30 °C and 3′ A-tailing for 30 min at 37 °C, followed by incubation at 70 °C for 5 min, and includes a clean-up and size selection step between end repair and 3′ A-tailing. The Kapa Hyper and NEBNext Ultra workflows include an enzyme mixture to perform end repair for 30 min at 20 °C, followed by 3′ A-tailing for 30 min at 65 °C.

    Techniques Used: Genome Wide, Polymerase Chain Reaction, Produced, Generated, Incubation, Selection

    11) Product Images from "Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA"

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-34079-2

    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    Figure Legend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Techniques Used: Labeling, Incubation, Negative Control

    Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.
    Figure Legend Snippet: Effect of end repair and 3′ A-tailing at high temperature on GC-bias in Illumina reads from PCR-free human DNA libraries. ( a ) Comparison of GC-bias curves in duplicate libraries prepared by immobilized enzymes with 3′ A-tailing performed at 37 °C (IM 37 °C -1 and IM 37 °C -2, in blue) or 65 °C (IM 65 °C -1 and IM 65 °C -2, in green) revealed a dramatic effect of 3′ A-tailing at high temperature on sequence coverage of the AT-rich regions from human DNA libraries. ( b ) GC-bias curves were generated from two sets of duplicate libraries produced using the soluble enzyme mixture PKT with (PKT purify-1 and PKT purify-2) or without (PKT-1 and PKT-2) a purification step between end repair and high temperature incubation (with Taq DNA pol added to the samples following purification). Although some bias against AT-rich regions can be attributed to the end repair step, the elevated temperature contributes to the majority of the dropouts in the AT-rich regions. ( c ) Shown are the GC-bias curves from 4 sets of duplicate libraries produced by the method of soluble enzymes. Two sets of the duplicate libraries were purified after end repair with PK mixture and then treated at 37 °C with Klenow Fragment (3′-5′ exo − ) (red, Klenow 37 °C-1 and Klenow 37 °C-2) or Taq DNA pol (blue, Taq 37 °C-1 and Taq 37 °C-2). The other two duplicate sets were prepared using the high temperature treatment protocol either with (green, Taq 65 °C-1 and Taq 65 °C-2) or without (orange, PKT-1 and PKT-2) a purification step between end repair with PKT and treatment with Taq DNA pol at 65 °C for 30 min. ( d ) Comparison of library yield of the three sets described above with or without (PKT on the left) a purification step between end repair and 3′ A-tailing indicates that purification caused substantial loss of library DNA.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Generated, Produced, Purification, Incubation

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.
    Figure Legend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 -benzylguanine (BG) moieties that specifically react with active site cysteine residues of SNAP-tag proteins, forming a stable covalent thioether bond 15 , 16 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Techniques Used: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.
    Figure Legend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c ) Processing AT-rich DNA by Taq DNA pol at elevated temperatures. During high temperature incubation, for example, at 65 °C or 70 °C, the ends of AT-rich DNA fragments melt into transient or predominant single-stranded structures. Taq DNA pol (red) can act on these DNA substrates by its polymerization and 5′ nuclease activities as previously described 34 , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Techniques Used: Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation, Activated Clotting Time Assay

    12) Product Images from "Deep learning image recognition enables efficient genome editing in zebrafish by automated injections"

    Article Title: Deep learning image recognition enables efficient genome editing in zebrafish by automated injections

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202377

    Average injection time required to obtain one positive genetically modified larva. Abbreviations: MO, slc45a2 morpholino; gRNA, slc45a2 gRNA/Cas9; DNA, Tol2 construct; Auto, automated injections; P1-4, four different experimentalists.
    Figure Legend Snippet: Average injection time required to obtain one positive genetically modified larva. Abbreviations: MO, slc45a2 morpholino; gRNA, slc45a2 gRNA/Cas9; DNA, Tol2 construct; Auto, automated injections; P1-4, four different experimentalists.

    Techniques Used: Injection, Genetically Modified, Construct

    13) Product Images from "CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants"

    Article Title: CRISPR-Mediated Knockout of Cybb in NSG Mice Establishes a Model of Chronic Granulomatous Disease for Human Stem-Cell Gene Therapy Transplants

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2017.005

    Cybb mutations identified in mice arising from clustered regularly interspaced short palindromic repeats (CRISPR) targeting of Cybb exon 1 or exon 3. ( A ) Cybb exon 1 deletions > 12 bp. Shown are a portion of the Cybb promoter and 5′ untranslated region (UTR), all of exon 1, and a portion of intron 1. The target site of the exon 1 CRISPR is also shown, as are the locations of larger deletions identified in mice, including the 235 bp deletion of the NSG.Cybb[KO] strain. ( B ) Cybb exon 3 deletions > 12 bp. Shown are a portion of the introns 2 and 3 and all of exon 3, with the target site of the exon 3 CRISPR and the locations of larger deletions identified in mice. ( C ) and ( D ) Small (≤12 bp) Cybb deletions identified in mice from CRISPR targeting of ( C ) exon 1 or ( D ) exon 3. Wild-type (wt) sequences for a portion of the target region are listed. Exon sequences are in uppercase, intron sequences are in lowercase, and CRISPR target sequences are underlined. For deletions matching those predicted to occur through the microhomology-mediated end joining (MMEJ) DNA repair pathway, the 2–3 bp microhomology sequences are listed.
    Figure Legend Snippet: Cybb mutations identified in mice arising from clustered regularly interspaced short palindromic repeats (CRISPR) targeting of Cybb exon 1 or exon 3. ( A ) Cybb exon 1 deletions > 12 bp. Shown are a portion of the Cybb promoter and 5′ untranslated region (UTR), all of exon 1, and a portion of intron 1. The target site of the exon 1 CRISPR is also shown, as are the locations of larger deletions identified in mice, including the 235 bp deletion of the NSG.Cybb[KO] strain. ( B ) Cybb exon 3 deletions > 12 bp. Shown are a portion of the introns 2 and 3 and all of exon 3, with the target site of the exon 3 CRISPR and the locations of larger deletions identified in mice. ( C ) and ( D ) Small (≤12 bp) Cybb deletions identified in mice from CRISPR targeting of ( C ) exon 1 or ( D ) exon 3. Wild-type (wt) sequences for a portion of the target region are listed. Exon sequences are in uppercase, intron sequences are in lowercase, and CRISPR target sequences are underlined. For deletions matching those predicted to occur through the microhomology-mediated end joining (MMEJ) DNA repair pathway, the 2–3 bp microhomology sequences are listed.

    Techniques Used: Mouse Assay, CRISPR

    14) Product Images from "Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens"

    Article Title: Cyclic Di-GMP Binding by an Assembly ATPase (PilB2) and Control of Type IV Pilin Polymerization in the Gram-Positive Pathogen Clostridium perfringens

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00034-17

    (A) TFP-associated genes in C. perfringens strain 13. The two sets of PilB/PilC-encoding genes are colored gray. (B) Phase-contrast and fluorescence imaging of YFP-PilB2 in strains HN13 (wild type; left) and WH2 ( pilC2 mutant; right). YFP-PilB2 appears as white spots in the cells. Note that some of the fluorescent foci are not at the poles. Bars = 5 μm. (C) Numbers of polar spots per cell for different strains of C. perfringens . The values shown are means and standard errors of the means (SEM). The fluorescent protein fusion used for each strain is shown in parentheses (pAH10 was used to express YFP-PilB2, and pAH12 was used for YFP-PilB1).
    Figure Legend Snippet: (A) TFP-associated genes in C. perfringens strain 13. The two sets of PilB/PilC-encoding genes are colored gray. (B) Phase-contrast and fluorescence imaging of YFP-PilB2 in strains HN13 (wild type; left) and WH2 ( pilC2 mutant; right). YFP-PilB2 appears as white spots in the cells. Note that some of the fluorescent foci are not at the poles. Bars = 5 μm. (C) Numbers of polar spots per cell for different strains of C. perfringens . The values shown are means and standard errors of the means (SEM). The fluorescent protein fusion used for each strain is shown in parentheses (pAH10 was used to express YFP-PilB2, and pAH12 was used for YFP-PilB1).

    Techniques Used: Fluorescence, Imaging, Mutagenesis

    15) Product Images from "Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid"

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    Journal: Nucleic Acids Research

    doi:

    N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.
    Figure Legend Snippet: N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Techniques Used: Activity Assay, High Performance Liquid Chromatography, Incubation, Labeling

    16) Product Images from "Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis"

    Article Title: Alternative Excision Repair of Ultraviolet B- and C-Induced DNA Damage in Dormant and Developing Spores of Bacillus subtilis

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01340-12

    (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .
    Figure Legend Snippet: (A to C) Levels of β-galactosidase from B. subtilis wild-type (A) and Δσ G (B) strains containing a ywjD-lacZ fusion and RT-PCR analysis of ywjD transcription (C). (A and B) B. subtilis strains PERM557 ( ywjD-lacZ ) (A) and PERM755 ( sigGΔ1 ywjD-lacZ ). (C) RNA samples (∼1 μg) isolated from a B. subtilis 168 DSM culture at the times indicated were processed for RT-PCR analysis as described in Materials and Methods. The arrowhead shows the size of the expected RT-PCR products. Lanes: M, DNA markers, 1-kb Plus ladder; Veg, logarithmic growth; T 0 , the time when the slopes of the logarithmic and stationary phases of growth intersected; T 1 to T 9 , times in hours after T 0 .

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Isolation

    17) Product Images from "Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA"

    Article Title: Optimization of enzymatic reaction conditions for generating representative pools of cDNA from small RNA

    Journal: RNA

    doi: 10.1261/rna.2242610

    Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.
    Figure Legend Snippet: Optimization of RNA 3′-end adapter ligation. ( A ) Temperature optimization. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapters (Linker) at different temperatures for either 2 or 18 h with 200 units of T4 Rnl2tr or without enzyme (−; input control). Ligation products were resolved and visualized by SYBR Gold staining. Ligation efficiency at varying temperatures is graphically represented as the mean ± SEM of four independent experiments. ( B ) Polyethylene glycol (PEG) as a ligation enhancer. Ligations were performed in the presence of varying concentrations of polyethylene glycol 8000 (PEG). Final concentrations in the reaction were 6.25%, 12.5%, and 25% (w/v). Ligation reactions were incubated for either 2 h or 18 h at 22°C or 16°C as indicated using 200 units of T4 Rnl2tr. (−) Indicates the absence of ligase. Ligation efficiency at varying concentrations of PEG 8000 is graphically represented as the mean ± SEM of three independent experiments. ( C ) Enzyme concentration. Ligations were performed using increasing amounts truncated T4 Rnl2tr (0, 10, 50, 100, 200, 500, 1000 units) in a reaction buffer containing 25% PEG 8000 (w/v) for 2 h at room temperature. Ligation efficiency using increasing amounts of enzyme are graphically represented as the mean ± SEM of three independent experiments.

    Techniques Used: Ligation, Staining, Incubation, Concentration Assay

    RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end attachment. ( A ) Comparison of optimized T4 Rnl2tr ligation to published ligation conditions. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends were ligated to pre-adenylated DNA adapter (AppLinker) using T4 Rnl2tr or T4 Rnl1 under different ligation conditions (conditions 1, 2, 3; detailed in Materials and Methods). Ligation products were resolved and visualized by SYBR Gold staining. ( B ) Quantification of ligation efficiency. Percent ligation refers to the amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Staining

    Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .
    Figure Legend Snippet: Ligation of adenylated adapters to cDNA 3′-ends. Pre-adenylated DNA oligonucleotides were ligated to synthetic double-stranded, partially double-stranded, or single-stranded oligonucleotides that mimic reaction products from reverse transcription of 3′-end ligated small RNAs. In the schematic representations of ligation inputs shown: (black lines) DNA; (gray lines) RNA; (star) IRDye 700. ( A ) Ligation of pre-adenylated DNA adapters to double-stranded reverse transcription products. Ligation products were separated by denaturing PAGE and visualized by IR fluorescence scanning. Ligation efficiency was determined as described in the Materials and Methods and is presented as the mean ± SEM of three independent experiments. Incubation and buffer conditions are detailed in the Materials and Methods section. ( B ) Ligation of pre-adenylated adapters to RNase H-treated reverse transcription products. The efficiency of ligation of pre-adenylated DNA adapters to RNase H-treated substrates is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A . ( C ) Ligation of pre-adenylated adapters to single-stranded DNA oligonucleotides. The efficiency of ligation of pre-adenylated DNA adapters to synthetic single-stranded DNA oligonucleotides is represented graphically as the mean ± SEM of three independent experiments. Ligase, buffer composition, and incubation conditions correspond to those in panel A .

    Techniques Used: Ligation, Polyacrylamide Gel Electrophoresis, Fluorescence, Incubation

    RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.
    Figure Legend Snippet: RNA 3′-end adapter ligation bias against 2′- O -methylated small RNA 3′-ends. Synthetic ssRNA oligonucleotides with either 2′-hydroxyl (OH) or 2′- O -methyl ( O -Me) 3′-ends and different 3′-terminal nucleotides (A, C, G, or U) were ligated to a pre-adenylated DNA adapter (AppLinker) using either T4 Rnl2tr or T4 Rnl1. Ligation products were resolved and visualized by SYBR Gold staining. Percent ligation refers to the relative amount of input RNA converted to ligated species as measured by densitometry. Data points represent the mean ± SEM; n = 3 experimental replicates.

    Techniques Used: Ligation, Methylation, Staining

    18) Product Images from "Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination"

    Article Title: Real-time single-molecule tethered particle motion experiments reveal the kinetics and mechanisms of Cre-mediated site-specific recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks274

    Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreY324F . ( A ) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel lox P sites in response to the addition of CreY324F. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. ( B ) The reaction scheme of the Cre-mediated site-specific recombination process in a 1267 bp DNA molecule containing parallel lox P sites. The initial substrate is a 1267 bp DNA molecule containing parallel lox P sites with an average BM value of 79.4 ± 7.7 nm. Two complexes are formed after the addition of Cre recombinase. The first complex is the synaptic complex, corresponding to the formation of a Cre tetramer with an average BM value of 47.5 ± 9.4 nm. The other complex is the abortive complex that failed to synapse within the duration of observations, corresponding to the association of Cre dimers with lox P sites, with an average BM value of 62.0 ± 4.2 nm. When 30 μl of 0.05% SDS was added after 30 min of incubation time, the reaction was stopped. Two different outcomes were observed: (i) a molecule that synapsed but lacked the strand-cleavage capability to complete recombination and returned to the original DNA substrate; and (ii) a molecule that failed to synapse within the duration of observations and eventually returned to the original DNA substrate. ( C ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 79.4 ± 7.7 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (62.0 ± 4.2 and 47.5 ± 9.4 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude after 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 42). The distribution of the dwell times between recombinase addition and the change in BM amplitude ( D ) to a value representing the abortive complex state and an association rate constant of (1.6 ± 0.2) × 10 4 M −1 s −1 ( R 2 = 0.96, n = 18) were obtained. ( F ) To a value that represents the synapse state, and an association rate constant of (1.1 ± 0.0) × 10 4 M −1 s −1 ( R 2 = 0.99, n = 64) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. ( E ) (i) The dwell times in the abortive complex state for DNA molecules containing parallel lox P sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [ R 2 = 0.98, τ 1 = (1.1 ± 0.1) × 10 −2 s −1 , n = 40]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [ R 2 = 0.98, k 1 = (3.2 ± 0.3) × 10 −2 s −1 , n = 189] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (6.3 ± 0.5) × 10 −2 s −1 , A 1 = 0.95; k 2 = (5.7 ± 0.5) × 10 −3 s −1 , A 2 = 0.05, n = 189]. The solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events that were observed. The error is within the 95% CL. (F) The mechanism through which bacteriophage P1 CreY324F mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel lox P sites. The empty circles represent Cre recombinases.
    Figure Legend Snippet: Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreY324F . ( A ) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel lox P sites in response to the addition of CreY324F. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. ( B ) The reaction scheme of the Cre-mediated site-specific recombination process in a 1267 bp DNA molecule containing parallel lox P sites. The initial substrate is a 1267 bp DNA molecule containing parallel lox P sites with an average BM value of 79.4 ± 7.7 nm. Two complexes are formed after the addition of Cre recombinase. The first complex is the synaptic complex, corresponding to the formation of a Cre tetramer with an average BM value of 47.5 ± 9.4 nm. The other complex is the abortive complex that failed to synapse within the duration of observations, corresponding to the association of Cre dimers with lox P sites, with an average BM value of 62.0 ± 4.2 nm. When 30 μl of 0.05% SDS was added after 30 min of incubation time, the reaction was stopped. Two different outcomes were observed: (i) a molecule that synapsed but lacked the strand-cleavage capability to complete recombination and returned to the original DNA substrate; and (ii) a molecule that failed to synapse within the duration of observations and eventually returned to the original DNA substrate. ( C ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 79.4 ± 7.7 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (62.0 ± 4.2 and 47.5 ± 9.4 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude after 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 42). The distribution of the dwell times between recombinase addition and the change in BM amplitude ( D ) to a value representing the abortive complex state and an association rate constant of (1.6 ± 0.2) × 10 4 M −1 s −1 ( R 2 = 0.96, n = 18) were obtained. ( F ) To a value that represents the synapse state, and an association rate constant of (1.1 ± 0.0) × 10 4 M −1 s −1 ( R 2 = 0.99, n = 64) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. ( E ) (i) The dwell times in the abortive complex state for DNA molecules containing parallel lox P sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [ R 2 = 0.98, τ 1 = (1.1 ± 0.1) × 10 −2 s −1 , n = 40]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [ R 2 = 0.98, k 1 = (3.2 ± 0.3) × 10 −2 s −1 , n = 189] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (6.3 ± 0.5) × 10 −2 s −1 , A 1 = 0.95; k 2 = (5.7 ± 0.5) × 10 −3 s −1 , A 2 = 0.05, n = 189]. The solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events that were observed. The error is within the 95% CL. (F) The mechanism through which bacteriophage P1 CreY324F mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel lox P sites. The empty circles represent Cre recombinases.

    Techniques Used: Incubation

    Complex formation and recombination events for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. ( A ) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel lox P sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. ( B ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time ( n = 43). ( C ) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. ( D ) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel lox P sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [ R 2 = 0.98, τ 1 = (4.0 ± 0.7) × 10 −3 s −1 , n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [ R 2 = 0.98, k 1 = (6.8 ± 0.8) × 10 −3 s −1 , n = 148] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (4.1 ± 1.9) × 10 −2 s −1 , A 1 = 0.90; k 2 = (2.6 ± 0.2) × 10 −3 s −1 , A 2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. ( E ) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel lox P sites. The empty circles r epresent Cre recombinases. The arrows on the DNA molecule indicate the parallel lox P sites.
    Figure Legend Snippet: Complex formation and recombination events for 1267 bp DNA molecules containing parallel loxP sites mediated by cleavage-deficient CreK201A. ( A ) (i)–(ii) A change in the BM amplitude of 1267 bp DNA molecules containing parallel lox P sites in response to the addition of CreK201A. (i) An example of a molecule that synapsed but failed to continue to the recombination process. (ii) An example of a molecule that failed to synapse within the duration of the observations. ( B ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, with an average value of 78.8 ± 10.2 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (61.0 ± 1.3 nm and 44.0 ± 12.0 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude at 30 min of incubation time. (iv) The distribution of the BM amplitude in response to the SDS challenge at 30 min of incubation time ( n = 43). ( C ) The distribution of the dwell times between recombinase addition and the change in the BM amplitude to (i) a value that represented the abortive complex state and an association rate constant of (2.0 ± 0.1) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 16) and to (ii) a value that represented the synapse state and an association rate constant of (1.5 ± 0.1) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 121) were obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules observed. ( D ) (i) The dwell times in the abortive complex state for the DNA molecules containing parallel lox P sites that failed to synapse during the observations were pooled and then fitted to a single exponential decay algorithm [ R 2 = 0.98, τ 1 = (4.0 ± 0.7) × 10 −3 s −1 , n = 7]. (ii) The distribution of the dwell times in the synapse state was fitted to either a single exponential decay algorithm [ R 2 = 0.98, k 1 = (6.8 ± 0.8) × 10 −3 s −1 , n = 148] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (4.1 ± 1.9) × 10 −2 s −1 , A 1 = 0.90; k 2 = (2.6 ± 0.2) × 10 −3 s −1 , A 2 = 0.10, n = 148]. Solid and dashed lines represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL. ( E ) The mechanism through which bacteriophage P1 CreK201A mediates site-specific recombination. The arrows on the DNA molecule indicate the parallel lox P sites. The empty circles r epresent Cre recombinases. The arrows on the DNA molecule indicate the parallel lox P sites.

    Techniques Used: Incubation

    Complex formation and recombination events of 1267 bp DNA molecules containing inverse loxP sites. ( A ) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse lox P sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. ( B ) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse lox P sites. The starting substrates are 1267 bp DNA molecules containing inverse lox P sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with lox P sites with an average BM value of 64.3 ± 3.6 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. ( C ) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude ( D ) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 18) were obtained. ( E ) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 81) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL.
    Figure Legend Snippet: Complex formation and recombination events of 1267 bp DNA molecules containing inverse loxP sites. ( A ) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing inverse lox P sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed and then returned to the original substrate, corresponding to either no reaction or completion of recombination. (ii) An example of a molecule that synapsed and was then trapped in a stable Holliday junction intermediate. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. ( B ) Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing inverse lox P sites. The starting substrates are 1267 bp DNA molecules containing inverse lox P sites and exhibit an average BM amplitude of 79.8 ± 7.5 nm. Two complexes are formed after interaction with Cre recombinase. The first complex is the synaptic complex, which corresponds to the formation of a Cre tetramer with an average BM value of 48.6 ± 10.2 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations and corresponds to the association of Cre dimers with lox P sites with an average BM value of 64.3 ± 3.6 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but either failed to complete recombination or formed the recombinant product after completion of the recombination process; (ii) a molecule that synapsed but was trapped in a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. ( C ) (i) The distribution of the BM amplitude before the addition of Cre recombinase showed an average value of 79.8 ± 7.5 nm, which is indicated with (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase (64.3 ± 3.6, 48.6 ± 10.2 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively). (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 101). The distribution of the dwell times between recombinase addition and the change in the BM amplitude ( D ) to a value representing the abortive complex state and an association rate constant of (8.1 ± 0.5) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 18) were obtained. ( E ) To a value that represents the synapse state, and an association rate constant of (6.4 ± 0.3) × 10 4 M −1 s −1 ( R 2 = 0.98, n = 81) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL.

    Techniques Used: Incubation, Recombinant

    Tethered particle motion experiment investigating the specific interaction between Cre recombinase and loxP sites . ( A ) Control experiment: the histogram of the BM amplitude was obtained using 1267 bp DNA molecules without lox P sites. The average BM value peaks at 81.6 ± 8.6 nm ( n = 53). ( B ) A histogram of the BM amplitude obtained from 1267 bp DNA molecules without a lox P site after a 1-h incubation with 200 nM Cre recombinase. The histogram was fitted to a two-Gaussian distribution that represented two conditions, in which the centers of the peaks occur at BM values of 81.2 ± 9.6 nm and 62.9 ± 11.5 nm for naked DNA and Cre-bound DNA, respectively ( n = 53). ( C ) The change in the BM amplitude of a single 1267 bp DNA molecule without a lox P site in response to the addition of Cre recombinase. The dashed line indicates the addition of Cre recombinase. ( D ) Control experiment: the histogram of the BM amplitude was obtained using 1267 bp DNA molecules containing one lox P site. The average BM value peaks at 80.5 ± 12.0 nm ( n = 85). ( E ) A histogram of the BM amplitude obtained from 1267 bp DNA molecules containing one lox P site after 1-h incubation with 200 nM Cre recombinase. The histogram was fitted to a two-Gaussian distribution that represented two conditions, in which the centers of the peaks occur at BM values of 80.5 ± 12.5 and 58.3 ± 9.4 nm for naked DNA and Cre-bound DNA, respectively ( n = 85). ( F ) The change in the BM amplitude of a single 1267 bp DNA molecule containing one lox P site in response to the addition of Cre recombinase. The dashed line indicates the addition of Cre recombinase. The cartoons shown below illustrate the experimental procedure. The white ball represents the streptavidin-labeled 200 nm polystyrene bead, the white spots represent the Cre recombinase molecules, the black curve represents the DNA molecules, the dashed arrowed line represents the Brownian motion amplitude, and the white line represents the lox P sequence. The error is the 95% CL.
    Figure Legend Snippet: Tethered particle motion experiment investigating the specific interaction between Cre recombinase and loxP sites . ( A ) Control experiment: the histogram of the BM amplitude was obtained using 1267 bp DNA molecules without lox P sites. The average BM value peaks at 81.6 ± 8.6 nm ( n = 53). ( B ) A histogram of the BM amplitude obtained from 1267 bp DNA molecules without a lox P site after a 1-h incubation with 200 nM Cre recombinase. The histogram was fitted to a two-Gaussian distribution that represented two conditions, in which the centers of the peaks occur at BM values of 81.2 ± 9.6 nm and 62.9 ± 11.5 nm for naked DNA and Cre-bound DNA, respectively ( n = 53). ( C ) The change in the BM amplitude of a single 1267 bp DNA molecule without a lox P site in response to the addition of Cre recombinase. The dashed line indicates the addition of Cre recombinase. ( D ) Control experiment: the histogram of the BM amplitude was obtained using 1267 bp DNA molecules containing one lox P site. The average BM value peaks at 80.5 ± 12.0 nm ( n = 85). ( E ) A histogram of the BM amplitude obtained from 1267 bp DNA molecules containing one lox P site after 1-h incubation with 200 nM Cre recombinase. The histogram was fitted to a two-Gaussian distribution that represented two conditions, in which the centers of the peaks occur at BM values of 80.5 ± 12.5 and 58.3 ± 9.4 nm for naked DNA and Cre-bound DNA, respectively ( n = 85). ( F ) The change in the BM amplitude of a single 1267 bp DNA molecule containing one lox P site in response to the addition of Cre recombinase. The dashed line indicates the addition of Cre recombinase. The cartoons shown below illustrate the experimental procedure. The white ball represents the streptavidin-labeled 200 nm polystyrene bead, the white spots represent the Cre recombinase molecules, the black curve represents the DNA molecules, the dashed arrowed line represents the Brownian motion amplitude, and the white line represents the lox P sequence. The error is the 95% CL.

    Techniques Used: Incubation, Labeling, Sequencing

    Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites . ( A ) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm ( n = 103). ( B ) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel lox P sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. ( C ). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel lox P sites. The initial substrate is the 1267 bp DNA molecule containing parallel lox P sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with lox P sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. ( D ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude ( E ) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 10 4 M −1 s −1 ( R 2 = 0.96, n = 16) were obtained. ( F ) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 10 4 M −1 s −1 ( R 2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. ( G ) BM time-trace of a 1267 bp DNA molecule containing parallel lox P sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. ( H ) The dwell times in the abortive complexes formed with the DNA molecules containing parallel lox P sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [ R 2 = 0.98, τ 1 = (5.2 ± 1.0) × 10 −3 s −1 , n = 26]. ( I ) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10 −1 s −1 ( n = 17, R 2 = 0.99), (6.6 ± 0.4) × 10 −1 s −1 ( n = 16, R 2 = 0.97) and (4.2 ± 0.7) × 10 −1 s −1 ( n = 40, R 2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.
    Figure Legend Snippet: Complex formation and recombination events of 1267 bp DNA molecules containing parallel loxP sites . ( A ) A histogram of the BM amplitude obtained from the 580 bp DNA molecules representing the recombinant products. The average BM value peaks at 43.3 ± 6.8 nm ( n = 103). ( B ) (i)–(iii) The change in the BM amplitude of the 1267 bp DNA molecules containing parallel lox P sites in response to the addition of Cre recombinase. (i) An example of a molecule that synapsed but failed to continue through the recombination process. (ii) An example of a molecule that synapsed and continued through the strand-cleavage step to the strand-migration step and then to the strand-ligation steps. (iii) An example of a molecule that failed to synapse within the duration of the observations. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern labels the average BM value of the expected recombinant excision product. ( C ). Reaction scheme of the Cre-mediated site-specific recombination process involving 1267 bp DNA molecules containing parallel lox P sites. The initial substrate is the 1267 bp DNA molecule containing parallel lox P sites and has an average BM amplitude of 80.5 ± 10.5 nm. Two complexes are formed after interaction with Cre recombinase. The first is the synaptic complex that corresponds to the formation of a Cre tetramer with an average BM value of 43.8 ± 8.7 nm. The other complex is the abortive complex that failed to synapse within the duration of the observations, corresponding to the association of Cre dimers with lox P sites with an average BM value of 59.3 ± 6.8 nm. The reaction was stopped when 30 μl of 0.05% SDS was added after 30 min of incubation time. Three different outcomes were observed: (i) a molecule that synapsed but failed to complete the recombination process; (ii) a molecule that synapsed and formed either the recombinant product or a stable Holliday junction intermediate; and (iii) a molecule that failed to synapse within the duration of the observations. ( D ) (i) The distribution of the BM amplitude before the addition of Cre recombinase, which shows an average value of 80.5 ± 10.5 nm, is indicated with the letter (c). (ii) The distribution of the BM amplitude in response to the addition of Cre recombinase [59.3 ± 6.8 nm and 43.8 ± 8.7 nm for the abortive complex state and the synapse state, marked with (b) and (a), respectively]. (iii) The distribution of the BM amplitude after 30 min of incubation time following the addition of Cre recombinase. (iv) The distribution of the BM amplitude in response to the SDS challenge after 30 min of incubation time ( n = 76). The distribution of the dwell times between recombinase addition and the change in the BM amplitude ( E ) to a value representing the abortive complex state and the association rate constant of (6.8 ± 0.4) × 10 4 M −1 s −1 ( R 2 = 0.96, n = 16) were obtained. ( F ) To a value representing the synapse state, and an association rate constant of (6.1 ± 0.6) × 10 4 M −1 s −1 ( R 2 = 0.99 n = 70) was obtained. The data were fitted to a single exponential decay algorithm (Origin 8.0). The N mentioned above is the number of molecules that were observed. The error is within the 95% CL. ( G ) BM time-trace of a 1267 bp DNA molecule containing parallel lox P sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and the addition of 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. (i) The BM time trace of a molecule that has successfully formed a synaptic complex following a short-lived presynapse state. (ii) The BM time trace of a molecule that failed to synapse and was trapped in the abortive complex state. For those molecules undergoing BM transitions from the original substrate to the synapse state, leading to an unsuccessful recombination, the short-lived presynapse state duration is classified as type a. For molecules that experience BM transitions from the synapse state to the original substrate, the short-lived presynapse state duration is classified as type b. For those molecules showing decreased BM, even after the SDS challenge, indicating the formation of a stable synapse state, the short-lived presynapse state duration is classified as type c. ( H ) The dwell times in the abortive complexes formed with the DNA molecules containing parallel lox P sites that failed to synapse during observation were pooled and then fitted to a single exponential decay [ R 2 = 0.98, τ 1 = (5.2 ± 1.0) × 10 −3 s −1 , n = 26]. ( I ) (i)–(iii) The dwell times in the short-lived presynapse state were pooled separately to build dwell-time histograms fitted to a single exponential decay algorithm with values of (7.7 ± 0.1) × 10 −1 s −1 ( n = 17, R 2 = 0.99), (6.6 ± 0.4) × 10 −1 s −1 ( n = 16, R 2 = 0.97) and (4.2 ± 0.7) × 10 −1 s −1 ( n = 40, R 2 = 0.99), representing the association rate constant of unstable synaptic complexes, the dissociation rate constant of presynaptic complexes and the association rate constant of correct synaptic complexes, respectively. The N mentioned above is the number of events observed. The error is within a 95% CL.

    Techniques Used: Recombinant, Migration, Ligation, Incubation

    Kinetic analysis of the dwell time in the synapse state. ( A ) The BM time-trace of a 1267 bp DNA molecule containing parallel lox P sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. ( B ) (i) The distribution of the dwell time in the synapse state, indicated by the double-arrowed line in the above time trace, for DNA molecules containing parallel lox P sites was pooled to construct a histogram, and the rate constants were obtained by fitting to either a single exponential decay algorithm [ R 2 = 0.91, k 1 = (4.4 ± 0.5) × 10 −3 s −1 , n = 118] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (2.7 ± 0.5) × 10 −2 s −1 , A 1 = 0.80; k 2 = (2.0 ± 0.1) × 10 −3 s −1 , A 2 = 0.20, n = 118]. Solid and dashed lines are used to represent single and bi-exponential fitting curves, respectively. (ii) The distribution of the dwell time in the synapse state, indicated by the double-arrowed line in the above time trace, for DNA molecules containing inverse lox P sites was pooled to construct a histogram, and the rate constants were obtained by fitting to either a single exponential decay algorithm [ R 2 = 0.91, k 1 = (6.5 ± 0.9) × 10 −3 s −1 , n = 178] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (2.7 ± 0.6) × 10 −2 s −1 , A 1 = 0.85; k 2 = (2.2 ± 0.2) × 10 −3 s −1 , A 2 = 0.15, n = 178]. Solid and dashed lines are used to represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL.
    Figure Legend Snippet: Kinetic analysis of the dwell time in the synapse state. ( A ) The BM time-trace of a 1267 bp DNA molecule containing parallel lox P sites in response to the addition of Cre recombinase. The dashed lines indicate the addition of Cre recombinase and 0.05% SDS after 30 min of incubation time. The bar with the punctuate pattern indicates the average BM value of the expected excision recombinant product. ( B ) (i) The distribution of the dwell time in the synapse state, indicated by the double-arrowed line in the above time trace, for DNA molecules containing parallel lox P sites was pooled to construct a histogram, and the rate constants were obtained by fitting to either a single exponential decay algorithm [ R 2 = 0.91, k 1 = (4.4 ± 0.5) × 10 −3 s −1 , n = 118] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (2.7 ± 0.5) × 10 −2 s −1 , A 1 = 0.80; k 2 = (2.0 ± 0.1) × 10 −3 s −1 , A 2 = 0.20, n = 118]. Solid and dashed lines are used to represent single and bi-exponential fitting curves, respectively. (ii) The distribution of the dwell time in the synapse state, indicated by the double-arrowed line in the above time trace, for DNA molecules containing inverse lox P sites was pooled to construct a histogram, and the rate constants were obtained by fitting to either a single exponential decay algorithm [ R 2 = 0.91, k 1 = (6.5 ± 0.9) × 10 −3 s −1 , n = 178] or a bi-exponential decay algorithm [ R 2 = 1.00, k 1 = (2.7 ± 0.6) × 10 −2 s −1 , A 1 = 0.85; k 2 = (2.2 ± 0.2) × 10 −3 s −1 , A 2 = 0.15, n = 178]. Solid and dashed lines are used to represent single and bi-exponential fitting curves, respectively. The N mentioned above is the number of events observed. The error is within the 95% CL.

    Techniques Used: Incubation, Recombinant, Construct

    19) Product Images from "Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response"

    Article Title: Leptospira interrogans serovar Copenhageni Harbors Two lexA Genes Involved in SOS Response

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076419

    Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).
    Figure Legend Snippet: Genomic and transcriptional organization of the lexA2 region. (A) Schematic representation of the lexA2 genomic region from L. interrogans serovar Copenhageni (upper) compared to the equivalent region of serovar Lai (lower). Arrows represent predicted genes and transcription orientation. Light grey arrows represent genes orthologous between genomes, dark grey genes that are specific to Copenhageni and black arrows indicate genes encoding transposases. The white arrows represent genes with truncated versions in Lai genome (traced arrows) by insertion of IS elements. Remnants of a phage integrase are indicated by a traced line. The numbered bars below the genes indicate the amplified fragments corresponding to the primer pairs used in the RT-PCR analyses. (B) RT-PCR reactions, using either genomic DNA (gDNA), RNA (RT-) or cDNA (RT+) as templates, and primers flanking intergenic regions. The numbers refer to the respective fragments shown in (A).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).
    Figure Legend Snippet: Genomic and transcriptional organization of the lexA1 region. (A) Schematic representation of the lexA1 genomic region. The arrows indicate the direction of transcription. The fragments amplified by the primer pairs used for the RT-PCR analysis are indicated by numbered lines below the genes. (B) Composite image of agarose gels from resulting RT-PCR reactions, using either genomic DNA (DNA), RNA (RT-) or cDNA (RT+) as templates. The numbers refer to the respective fragments shown in (A).

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction

    20) Product Images from "Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion"

    Article Title: Homologous Recombination Occurs in Entamoeba and Is Enhanced during Growth Stress and Stage Conversion

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0074465

    Demonstration of HR during growth stress in E. histolytica and encystation in E. invadens . Cells were transfected with the inverted repeat construct (Fig. 2). HR was demonstrated by measuring densitometrically the amplicon obtained with primer pair P2/P3 compared with primer pair P1/P2, by Southern analysis with LUC probe. Amplicon obtained by primer set P2/P3 was sequenced for confirmation of recombination (Fig. S2 in file S1 ). R.R.: Relative Recombination (ratio of amplicon intensity with P2/P3 versus P1/P2, with respect to normal trophozoites (N) as 1). Recombination was checked in different stress conditions i.e. (A) serum starvation; (B) heat stress; (C) oxygen stress and (D) UV irradiation with UV-C light (150 J/m 2 ) for 8 sec followed by incubation in fresh medium for indicated time periods [21] ; (E) E. invadens cells transferred to encystation medium and (F) excystation in fresh medium. UT: DNA from un-transfected cells; P: DNA of LUC plasmid. Full-length blots are presented in Fig. S4 in file S1 .
    Figure Legend Snippet: Demonstration of HR during growth stress in E. histolytica and encystation in E. invadens . Cells were transfected with the inverted repeat construct (Fig. 2). HR was demonstrated by measuring densitometrically the amplicon obtained with primer pair P2/P3 compared with primer pair P1/P2, by Southern analysis with LUC probe. Amplicon obtained by primer set P2/P3 was sequenced for confirmation of recombination (Fig. S2 in file S1 ). R.R.: Relative Recombination (ratio of amplicon intensity with P2/P3 versus P1/P2, with respect to normal trophozoites (N) as 1). Recombination was checked in different stress conditions i.e. (A) serum starvation; (B) heat stress; (C) oxygen stress and (D) UV irradiation with UV-C light (150 J/m 2 ) for 8 sec followed by incubation in fresh medium for indicated time periods [21] ; (E) E. invadens cells transferred to encystation medium and (F) excystation in fresh medium. UT: DNA from un-transfected cells; P: DNA of LUC plasmid. Full-length blots are presented in Fig. S4 in file S1 .

    Techniques Used: Transfection, Construct, Amplification, Irradiation, Size-exclusion Chromatography, Incubation, Plasmid Preparation

    21) Product Images from "Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome"

    Article Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis reveals the structural complexity of the mammalian mitochondrial genome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1324

    Separation of mtDNA into major topoisomers: catenanes, relaxed circles, linear molecules and supercoiled circles. ( A ) Treatment of total cellular DNA isolated from MEFs with BglII and topoisomerase I (Topo I). Untreated control (untr.) is shown for reference. Additional structures present after BglII digest are indicated by asterisks. ( B ) Treatment of total cellular DNA isolated from C2C12 myoblasts with topoisomerases I, II, IV and gyrase. ( C ) Treatment of total cellular DNA isolated from C2C12 myoblasts with S1 nuclease and E. coli exonuclease I. All gels were probed by Southern blot with COXI mtDNA sequence.
    Figure Legend Snippet: Separation of mtDNA into major topoisomers: catenanes, relaxed circles, linear molecules and supercoiled circles. ( A ) Treatment of total cellular DNA isolated from MEFs with BglII and topoisomerase I (Topo I). Untreated control (untr.) is shown for reference. Additional structures present after BglII digest are indicated by asterisks. ( B ) Treatment of total cellular DNA isolated from C2C12 myoblasts with topoisomerases I, II, IV and gyrase. ( C ) Treatment of total cellular DNA isolated from C2C12 myoblasts with S1 nuclease and E. coli exonuclease I. All gels were probed by Southern blot with COXI mtDNA sequence.

    Techniques Used: Isolation, Southern Blot, Sequencing

    22) Product Images from "CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis"

    Article Title: CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erp309

    Identification of cwinv4 mutants. Two independent Arabidopsis T-DNA mutant lines ( Alonso et al. , 2003 ), cwinv4-1 (SALK_130163) and cwinv4-2 (SALK_017466C), were obtained from the Arabidopsis Biological Resource Centre and genotyped to obtain homozygous mutant plants. The relative locations of each T-DNA insertion are shown in (A). RT-PCR, performed on RNA isolated from whole flowers, demonstrated that cwinv4-1 is a null mutant, and that AtCWINV4 is expressed significantly lower in cwinv4-2 flowers than in wild-type (B).
    Figure Legend Snippet: Identification of cwinv4 mutants. Two independent Arabidopsis T-DNA mutant lines ( Alonso et al. , 2003 ), cwinv4-1 (SALK_130163) and cwinv4-2 (SALK_017466C), were obtained from the Arabidopsis Biological Resource Centre and genotyped to obtain homozygous mutant plants. The relative locations of each T-DNA insertion are shown in (A). RT-PCR, performed on RNA isolated from whole flowers, demonstrated that cwinv4-1 is a null mutant, and that AtCWINV4 is expressed significantly lower in cwinv4-2 flowers than in wild-type (B).

    Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Isolation

    23) Product Images from "Characterization of the Type III restriction endonuclease PstII from Providencia stuartii"

    Article Title: Characterization of the Type III restriction endonuclease PstII from Providencia stuartii

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki787

    Identification of the PstII methylation site. ( A ) Oligonucleotides were synthesized and annealed to generate non-specific and specific DNA substrates. Pre-modified sequences were generated by synthesizing oligonucleotides with an N6 methyl deoxyadenosine at either position 1, 2 or 3. ( B ) Scintillation counting of oligonucleotide substrates following incubation with PstII and [ 3 H-methyl]-AdoMet. See main text for full details.
    Figure Legend Snippet: Identification of the PstII methylation site. ( A ) Oligonucleotides were synthesized and annealed to generate non-specific and specific DNA substrates. Pre-modified sequences were generated by synthesizing oligonucleotides with an N6 methyl deoxyadenosine at either position 1, 2 or 3. ( B ) Scintillation counting of oligonucleotide substrates following incubation with PstII and [ 3 H-methyl]-AdoMet. See main text for full details.

    Techniques Used: Methylation, Synthesized, Modification, Generated, Incubation

    Rate of DNA cleavage by the PstII restriction endonuclease. pLJP11b (5 nM) was incubated for various times with saturating PstII and 4 mM ATP at 37°C (see main text for full details). Where indicated AdoMet was included at 100 μM. The CCC substrate (squares) was separated from the OC intermediate (triangles) and FLL product (circles) by agarose gel electrophoresis (data not shown). Fragments were excised and the percentages of the 3 H-labelled DNA fragments quantified by scintillation counting.
    Figure Legend Snippet: Rate of DNA cleavage by the PstII restriction endonuclease. pLJP11b (5 nM) was incubated for various times with saturating PstII and 4 mM ATP at 37°C (see main text for full details). Where indicated AdoMet was included at 100 μM. The CCC substrate (squares) was separated from the OC intermediate (triangles) and FLL product (circles) by agarose gel electrophoresis (data not shown). Fragments were excised and the percentages of the 3 H-labelled DNA fragments quantified by scintillation counting.

    Techniques Used: Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis

    DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.
    Figure Legend Snippet: DNA sequences and substrates for the PstII restriction enzyme. ( A ) Recognition sequences for PstII and EcoP15I. The adenine residue methylated is underlined. The non-specific cleavage loci are indicated by arrows. The arbitrary direction of the sites is indicated by the arrowhead (black/white) and is set by the location of the cleavage site relative to the recognition site. ( B ) DNA substrates used in PstII assays with total sizes in brackets (bp). Distances between sites represent nucleotide spacings not including the base pairs in the recognition sequence. Sites with different flanking sequences are indicated by ‘1’ or ‘2’ (see Figure 3 ). ( C ) Cleavage of DNA substrates from (B) with PstII as indicated (see main text for more details). The resulting fragments were separated by agarose gel electrophoresis. The locations of the intact plasmid and cleavage products are indicated [note that the plasmids differ in size, (B)]. For LinHtH, the cleavage of either site 1 or 2 produces a characteristic pair of DNA fragments.

    Techniques Used: Methylation, Sequencing, Agarose Gel Electrophoresis, Plasmid Preparation

    The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.
    Figure Legend Snippet: The Type III enzymes EcoP15I and PstII cannot mutually activate cleavage of T7 coliphage DNA. ( A ) Representative schematic (not to scale) of the relative orientation of EcoP15I and PstII sites in lambda (λ) and T7 phage genomic DNA. Site orientations (arrowheads) are defined as in Figure 2A . ( B ) Cleavage of λ and T7 genomic DNA by mixtures of Type III enzymes. 500 ng of λ or T7 phage DNA was mixed with 50 nM EcoP15I and/or 129 nM PstII mixture as shown in the presence of 4 mM ATP. Where indicated AdoMet was added to 100 μM. Following incubation for 1 h at 37°C, substrate and products were separated by agarose gel electrophoresis.

    Techniques Used: Incubation, Agarose Gel Electrophoresis

    Identification of the PstII cleavage loci using four sites with different flanking sequences. ( A ) Sequences of sites 1 and 2 from pLJP11b ( Figure 2B ), site 3 from pLJP12c and site 4 from pLJP12d. PstII recognition sequence in bold, shared flanking sequences underlined. Cleavage loci are indicated by an arrow. ( B ) Representative denaturing PAGE (site 2) showing separation of primer extension products following PstII cleavage. See main text for full details. A 32 P-labelled DNA generated by single round primer extension was cut with PstII to give a labelled product as shown (lines represent DNA, arrowhead represents PstII site as in Figure 2A ). Sample ‘−’ was then run directly on the gel, whilst sample ‘+’ was first treated with Klenow polymerase. Sizes of the resulting fragments were compared to sequencing reactions produced using the same primer. For presentation purposes the brightness and contrast of the digital images were differentially adjusted using a linear intensity scale to increase the relative contrast of the labelled fragments.
    Figure Legend Snippet: Identification of the PstII cleavage loci using four sites with different flanking sequences. ( A ) Sequences of sites 1 and 2 from pLJP11b ( Figure 2B ), site 3 from pLJP12c and site 4 from pLJP12d. PstII recognition sequence in bold, shared flanking sequences underlined. Cleavage loci are indicated by an arrow. ( B ) Representative denaturing PAGE (site 2) showing separation of primer extension products following PstII cleavage. See main text for full details. A 32 P-labelled DNA generated by single round primer extension was cut with PstII to give a labelled product as shown (lines represent DNA, arrowhead represents PstII site as in Figure 2A ). Sample ‘−’ was then run directly on the gel, whilst sample ‘+’ was first treated with Klenow polymerase. Sizes of the resulting fragments were compared to sequencing reactions produced using the same primer. For presentation purposes the brightness and contrast of the digital images were differentially adjusted using a linear intensity scale to increase the relative contrast of the labelled fragments.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Generated, Produced

    Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.
    Figure Legend Snippet: Nucleotide requirement and NTPase activity of PstII. ( A ) Comparison of nucleotide usage of EcoP15I and PstII. A total of 5 nM substrate (pMDS34a or pLJP11b) was incubated with saturating enzyme in the presence of 4 mM nucleotide as shown for 1 h at 37°C. DNA substrates (CCC, dimer, OC) and product fragments [OC, FLL, ( 2 )] were then separated by agarose gel electrohporesis. Two cleavage produces two linear fragements, the smaller of which was not resolved on these gels. ( B ) Apparent binding efficiency of ATP, CTP and GTP. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and increasing concentrations of NTP as indicated for 1 h at 37°C. The substrate and product fragments were separated by agarose gel electrophoresis and quantified by scintillation. The appearance of FLL product is shown. ( C ) Effect of nucleotide identity upon rate of cleavage. PstII mixture (129 nM) was incubated with 5 nM pLJP11b and 4 mM NTP as indicated at 37°C. Aliquots were removed from the reactions and quenched at the timepoints indicated and the percentage of FLL product determined as in (B). Nucleotide hydrolysis of PstII measured using an NADH coupled assay [see Materials and Methods, ( 27 )] ( D ). PstII mixture (129 nM) was incubated with 1 nM DNA (pSKfokI or pAT153) and NTPs as indicated at 37°C and the change in A 340 measured over 1 h. The site-specific rate was obtained from the difference between the non-specific (pSKfokI) and specific (pAT153) rates (Materials and Methods). Error bars represent the standard error of two repeat experiments. ( E ) PstII mixture (129 nM) was incubated for 4 s with 5 nM pLJP11b, 4 mM ATP and increasing concentrations of UTP as indicted. The proportion of FLL DNA was determined as above.

    Techniques Used: Activity Assay, Incubation, Countercurrent Chromatography, Agarose Gel Electrophoresis, Binding Assay

    24) Product Images from "Direct detection of methylation in genomic DNA"

    Article Title: Direct detection of methylation in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gni121

    Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.
    Figure Legend Snippet: Differences between G signals complementary to unmethylated C residues, N 4 -methylcytosine residues and 5-methylcytosine residues using the DYEnamic ET Terminator Kit. A1: untreated pUC19 DNA; A2: M.BamHI N 4 -cytosine methylated pUC19DNA; A3: trace difference between A2 and A1. B1: untreated pUC19DNA; B2: M.HaeIII 5-cytosine methylated pUC19 DNA; B3: trace difference between B2 and B1. The G residues complementary to the methylated cytosines are boxed. N 4 -methylcytosine results in an increase in the complementary G signal, whereas 5-methylcytosine results in a decrease in the complementary G signal.

    Techniques Used: Methylation

    25) Product Images from "DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation"

    Article Title: DISSECT Method Using PNA-LNA Clamp Improves Detection of EGFR T790m Mutation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0067782

    Comparison of PNA-LNA sensitivity of detection for the T790M EGFR mutation. (A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.
    Figure Legend Snippet: Comparison of PNA-LNA sensitivity of detection for the T790M EGFR mutation. (A) Real-time PCR plot represents serially diluted PCR product with T790M mutation (1%, 0.1% and 0.01%) into amplified product from wild type DNA, by conventional PNA-LNA PCR. (A, right) Corresponding plot of the log concentration of T790M mutant DNA versus threshold cycle number. Graph shows mutant T790M serial dilution following conventional PNA-LNA PCR. (B) Real-time PCR plot shows an increased level of detection when PNA-LNA is applied to a sample that has undergone two rounds of mutant enrichment by DISSECT. (B, right) Corresponding graph shows mutant T790M serial dilution following PNA-LNA PCR after DISSECT.

    Techniques Used: Mutagenesis, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Concentration Assay, Serial Dilution

    T790M EGFR mutant enrichment analysis by HRM and Sanger Sequencing. (A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M EGFR mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C > T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C > T.
    Figure Legend Snippet: T790M EGFR mutant enrichment analysis by HRM and Sanger Sequencing. (A) HRM analysis plots and Sanger Sequencing chromatograms of serial dilutions of T790M EGFR mutations versus wild type DNA following conventional PCR. (B) The detection after mutant enrichment by conventional PNA-LNA PCR using an initial mutant abundance of 1% and 0.1% T790M, c.2369C > T. (C) Improved detection after mutant enrichment from two rounds of DISSECT followed by conventional PNA-LNA PCR from an initial mutant abundance of 1% and 0.01% T790M, c.2369C > T.

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction

    26) Product Images from "A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA"

    Article Title: A Method for Selectively Enriching Microbial DNA from Contaminating Vertebrate Host DNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0076096

    MBD-Fc enriches E. coli DNA from mixed E. coli and human DNA (IMR-90) samples. Graphs showing the percentage of mapped reads from Ion Torrent PGM experiments to either the E. coli MG1655 or human hg19 reference genome from libraries made with different ratios of human to E. coli DNA. The ratio between E. coli to human DNA in the premixed samples is indicated above the figure. “Unenriched” refers to untreated, control mixtures. “Bound” indicates DNA that remained bound to MBD-Fc beads and “Enriched” corresponds to unbound DNA remaining in the supernatant.
    Figure Legend Snippet: MBD-Fc enriches E. coli DNA from mixed E. coli and human DNA (IMR-90) samples. Graphs showing the percentage of mapped reads from Ion Torrent PGM experiments to either the E. coli MG1655 or human hg19 reference genome from libraries made with different ratios of human to E. coli DNA. The ratio between E. coli to human DNA in the premixed samples is indicated above the figure. “Unenriched” refers to untreated, control mixtures. “Bound” indicates DNA that remained bound to MBD-Fc beads and “Enriched” corresponds to unbound DNA remaining in the supernatant.

    Techniques Used:

    27) Product Images from "Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing"

    Article Title: Genetic Variation of Human Papillomavirus Type 16 in Individual Clinical Specimens Revealed by Deep Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0080583

    Amplification of full-length HPV16 genomes by full-circle PCR. (A) PCR was performed with PrimeSTAR ® GXL DNA polymerase and HPV16-specific primer-pairs as indicated. The amounts of HPV16/pUC19 used for the PCR template are also indicated above. The PCR products were analyzed by agarose gel electrophoresis. M, DNA size markers. (B) Scheme for full-circle PCR. PrimeSTAR ® GXL DNA polymerase generates short and long DNA products with primer-pair 1742F/1873R. (C) Full-circle PCR with DNA extracted from W12 cells, clone 20863 (high-copy HPV16 episomes) (lane 2) and clone 20850 (low-copy HPV16 episomes) (lane 3). M, DNA size marker (lanes 1) (D) Full-circle PCR using DNA isolated from 7 clinical specimens: 5 LSIL (lanes 2 to 6), and 2 ICC (lanes 7 and 8). M, DNA size marker (lanes 1).
    Figure Legend Snippet: Amplification of full-length HPV16 genomes by full-circle PCR. (A) PCR was performed with PrimeSTAR ® GXL DNA polymerase and HPV16-specific primer-pairs as indicated. The amounts of HPV16/pUC19 used for the PCR template are also indicated above. The PCR products were analyzed by agarose gel electrophoresis. M, DNA size markers. (B) Scheme for full-circle PCR. PrimeSTAR ® GXL DNA polymerase generates short and long DNA products with primer-pair 1742F/1873R. (C) Full-circle PCR with DNA extracted from W12 cells, clone 20863 (high-copy HPV16 episomes) (lane 2) and clone 20850 (low-copy HPV16 episomes) (lane 3). M, DNA size marker (lanes 1) (D) Full-circle PCR using DNA isolated from 7 clinical specimens: 5 LSIL (lanes 2 to 6), and 2 ICC (lanes 7 and 8). M, DNA size marker (lanes 1).

    Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Isolation, Immunocytochemistry

    28) Product Images from "A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements"

    Article Title: A highly sensitive in vivo footprinting technique for condition-dependent identification of cis elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt883

    Schematic presentation of the workflow and generation of final data. The main steps of the software-based, high-throughput in vivo footprinting method comprise growing/incubating the microorganism under conditions to be investigated (e.g. inducing conditions), in vivo DNA methylation using e.g. DMS, DNA extraction, DNA cleavage by e.g. HCl followed by LM-PCR and CGE. A subset of CGE analyses results to be compared (raw data) are submitted to electronic data analysis using the ivFAST software for generation of the results displayed as final heatmap (processed data output). The steps of processing the data by the ivFAST software can be inferred from the flowchart (for more details see the ivFAST manual). Heatmap: x -axis gives the analysed DNA sequence; y -axis shows which samples are referred to each other (e.g. G/ND means ‘glucose repressing conditions referred to naked DNA’); only signals that are statistically different are considered; protected bases are highlighted in red shades and hypersensitive bases are highlighted in blue shades; 1.1- to 1.3-fold difference between compared conditions is shown in light shaded colour, 1.3- to 1.5-fold difference between compared conditions is shown in middle shaded colour and > 1.5-fold difference between compared conditions is shown in dark shaded colour.
    Figure Legend Snippet: Schematic presentation of the workflow and generation of final data. The main steps of the software-based, high-throughput in vivo footprinting method comprise growing/incubating the microorganism under conditions to be investigated (e.g. inducing conditions), in vivo DNA methylation using e.g. DMS, DNA extraction, DNA cleavage by e.g. HCl followed by LM-PCR and CGE. A subset of CGE analyses results to be compared (raw data) are submitted to electronic data analysis using the ivFAST software for generation of the results displayed as final heatmap (processed data output). The steps of processing the data by the ivFAST software can be inferred from the flowchart (for more details see the ivFAST manual). Heatmap: x -axis gives the analysed DNA sequence; y -axis shows which samples are referred to each other (e.g. G/ND means ‘glucose repressing conditions referred to naked DNA’); only signals that are statistically different are considered; protected bases are highlighted in red shades and hypersensitive bases are highlighted in blue shades; 1.1- to 1.3-fold difference between compared conditions is shown in light shaded colour, 1.3- to 1.5-fold difference between compared conditions is shown in middle shaded colour and > 1.5-fold difference between compared conditions is shown in dark shaded colour.

    Techniques Used: Software, High Throughput Screening Assay, In Vivo, Footprinting, DNA Methylation Assay, DNA Extraction, Polymerase Chain Reaction, Sequencing

    29) Product Images from "In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice"

    Article Title: In Vivo Expression Technology Identifies a Novel Virulence Factor Critical for Borrelia burgdorferi Persistence in Mice

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003567

    Spirochetes lacking bbk46 retain seroreactivity in mice. Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46 :: flaB p - aadA /pBSV2G (Δ bbk46 /vector) and bbk46 :: flaB p - aadA /pBSV2G- bbk46 (Δ bbk46 / bbk46 + ) at a dose of 1×10 4 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.
    Figure Legend Snippet: Spirochetes lacking bbk46 retain seroreactivity in mice. Immunoblot analysis of sera collected three weeks post inoculation from groups of five C3H/HeN mice inoculated with clone A3-68ΔBBE02 (WT), bbk46 :: flaB p - aadA /pBSV2G (Δ bbk46 /vector) and bbk46 :: flaB p - aadA /pBSV2G- bbk46 (Δ bbk46 / bbk46 + ) at a dose of 1×10 4 spirochetes per mouse. (A) Total protein lysate from B. burgdorferi clone B31 A3 was probed with the serum from each individual mouse (1–5). (B) Purified recombinant GST-OspC protein was probed with pooled sera from the five mice in each infection group or αOspC polyclonal antibodies. The positions of markers to the left of the panel depict protein standard molecular masses in kilodaltons.

    Techniques Used: Mouse Assay, Plasmid Preparation, Purification, Recombinant, Infection

    Generation of the Δ bbk46 mutant and genetic complemented clones in B. burgdorferi . (A) Schematic representation of the wild-type (WT) and Δ bbk46 loci on lp36. The sequence of the entire bbk46 open reading frame was replaced with a flaB p - aadA antibiotic resistance cassette [37] , [82] . Locations of primers for analysis of the mutant clones are indicated with small arrows and labels P7–P12, P19 and P20. Primer sequences are listed in Table 5 . (B) PCR analysis of the Δ bbk46 mutant clone. Genomic DNA isolated from WT and Δ bbk46/ vector spirochetes served as the template DNA for PCR analyses. DNA templates are indicated across the bottom of the gel image. The primer pairs used to amplify specific DNA sequences are indicated at the top of the gel image and correspond to target sequences as shown in A. Migration of the DNA ladder in base pairs is shown to the left of each image. (C) In vitro growth analysis of mutant clones. A3-68ΔBBE02 (WT), bbk46 :: flaB p - aadA /pBSV2G (Δ bbk46 /vector) and bbk46 :: flaB p - aadA /pBSV2G- bbk46 (Δ bbk46 / bbk46 + ) spirochetes were inoculated in triplicate at a density of 1×10 5 spirochetes/ml in 5 ml of BSKII medium. Spirochete densities were determined every 24 hours under dark field microscopy using a Petroff-Hausser chamber over the course of 96 hours. The data are represented as the number of spirochetes per ml over time (hours) and is expressed as the average of 3 biological replicates. Error bars indicate the standard deviation from the mean.
    Figure Legend Snippet: Generation of the Δ bbk46 mutant and genetic complemented clones in B. burgdorferi . (A) Schematic representation of the wild-type (WT) and Δ bbk46 loci on lp36. The sequence of the entire bbk46 open reading frame was replaced with a flaB p - aadA antibiotic resistance cassette [37] , [82] . Locations of primers for analysis of the mutant clones are indicated with small arrows and labels P7–P12, P19 and P20. Primer sequences are listed in Table 5 . (B) PCR analysis of the Δ bbk46 mutant clone. Genomic DNA isolated from WT and Δ bbk46/ vector spirochetes served as the template DNA for PCR analyses. DNA templates are indicated across the bottom of the gel image. The primer pairs used to amplify specific DNA sequences are indicated at the top of the gel image and correspond to target sequences as shown in A. Migration of the DNA ladder in base pairs is shown to the left of each image. (C) In vitro growth analysis of mutant clones. A3-68ΔBBE02 (WT), bbk46 :: flaB p - aadA /pBSV2G (Δ bbk46 /vector) and bbk46 :: flaB p - aadA /pBSV2G- bbk46 (Δ bbk46 / bbk46 + ) spirochetes were inoculated in triplicate at a density of 1×10 5 spirochetes/ml in 5 ml of BSKII medium. Spirochete densities were determined every 24 hours under dark field microscopy using a Petroff-Hausser chamber over the course of 96 hours. The data are represented as the number of spirochetes per ml over time (hours) and is expressed as the average of 3 biological replicates. Error bars indicate the standard deviation from the mean.

    Techniques Used: Mutagenesis, Clone Assay, Sequencing, Polymerase Chain Reaction, Isolation, Plasmid Preparation, Migration, In Vitro, Microscopy, Standard Deviation

    30) Product Images from "Deficiency in Repair of the Mitochondrial Genome Sensitizes Proliferating Myoblasts to Oxidative Damage"

    Article Title: Deficiency in Repair of the Mitochondrial Genome Sensitizes Proliferating Myoblasts to Oxidative Damage

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0075201

    Mitochondrial genome of myoblasts is highly sensitive to oxidant-induced damage. ( A ) The amount of ROS generated by 0.005, 0.05 and 0.5 U/ml of GOx detected by fluorescence of DCF at 0, 5, 15, 30 and 60 min in cultured myoblasts and myotubes. Integrity of the nuclear ( B ) and mitochondrial ( C ) genomes of myoblasts and myotubes cultured for 1 h in various concentrations of GOx analyzed by amplification of 9kb and 10kb of nuclear- and mitochondrial-specific DNA fragments, respectively, by long-amplicon (LA)-PCR technique. ( D ) Mitochondrial genome copy number was analyzed by amplification of the 117bp mitochondrial genome-specific DNA fragment. The mitochondrial genome copy number in UT myoblasts and myotubes were set as 1. The graphs are based on PCR amplification with three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). The integrity in untreated (UT) control myoblasts was set as 1. The integrity in UT myotubes is shown relative to UT myoblasts or set as 1. Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05 relative to UT controls.
    Figure Legend Snippet: Mitochondrial genome of myoblasts is highly sensitive to oxidant-induced damage. ( A ) The amount of ROS generated by 0.005, 0.05 and 0.5 U/ml of GOx detected by fluorescence of DCF at 0, 5, 15, 30 and 60 min in cultured myoblasts and myotubes. Integrity of the nuclear ( B ) and mitochondrial ( C ) genomes of myoblasts and myotubes cultured for 1 h in various concentrations of GOx analyzed by amplification of 9kb and 10kb of nuclear- and mitochondrial-specific DNA fragments, respectively, by long-amplicon (LA)-PCR technique. ( D ) Mitochondrial genome copy number was analyzed by amplification of the 117bp mitochondrial genome-specific DNA fragment. The mitochondrial genome copy number in UT myoblasts and myotubes were set as 1. The graphs are based on PCR amplification with three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). The integrity in untreated (UT) control myoblasts was set as 1. The integrity in UT myotubes is shown relative to UT myoblasts or set as 1. Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05 relative to UT controls.

    Techniques Used: Generated, Fluorescence, Cell Culture, Amplification, Polymerase Chain Reaction, Isolation

    Mitochondrial extracts of myoblasts accumulate DNA repair intermediates. ( A ) Total repair synthesis activity of tetrahydrofuran (THF) containing oligo3 duplex in the mitochondrial extract of myoblasts and myotubes. Schematic representation of repair reaction is shown above the radiogram. Repair efficiency is based on analysis of at least three independently isolated mitochondrial extracts for each cell type. Activity in the mitochondrial extracts of myoblasts was set as 1. * indicates p ≤ 0.05. ( B ) Repair efficiency of the mitochondrial genome of myoblasts and myotubes after GOx treatment was monitored by amplification of a 10kb mitochondrial-specific DNA fragment by LA-PCR. The level of integrity in UT myoblasts and myotubes was set as 1. ( C ) The relative number of mitochondrial genomes in myoblasts and myotubes was based on PCR amplification of 117bp mitochondrialDNA-specific fragment. The graphs represent PCR amplification of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. P, final repair product; INT, repair intermediates; UT, untreated control.
    Figure Legend Snippet: Mitochondrial extracts of myoblasts accumulate DNA repair intermediates. ( A ) Total repair synthesis activity of tetrahydrofuran (THF) containing oligo3 duplex in the mitochondrial extract of myoblasts and myotubes. Schematic representation of repair reaction is shown above the radiogram. Repair efficiency is based on analysis of at least three independently isolated mitochondrial extracts for each cell type. Activity in the mitochondrial extracts of myoblasts was set as 1. * indicates p ≤ 0.05. ( B ) Repair efficiency of the mitochondrial genome of myoblasts and myotubes after GOx treatment was monitored by amplification of a 10kb mitochondrial-specific DNA fragment by LA-PCR. The level of integrity in UT myoblasts and myotubes was set as 1. ( C ) The relative number of mitochondrial genomes in myoblasts and myotubes was based on PCR amplification of 117bp mitochondrialDNA-specific fragment. The graphs represent PCR amplification of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. P, final repair product; INT, repair intermediates; UT, untreated control.

    Techniques Used: Activity Assay, Isolation, Amplification, Polymerase Chain Reaction

    Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of EXOG-FLAG-tagged was monitored by Western analysis with FLAG-HRP conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.
    Figure Legend Snippet: Ectopic expression of EXOG increases resistance to oxidant-induced DNA damage in myoblasts. ( A ) The expression level of EXOG-FLAG-tagged was monitored by Western analysis with FLAG-HRP conjugated antibody. ( B ) The integrity of the mitochondrial genome in myoblasts and myotubes transfected with vector or EXOG expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity in UT control (empty vector transfected) myoblasts and myotubes was set as 1. ( C ) The integrity of the mitochondrial genome of the myoblasts transfected with vector or mitochondrial specific OGG1 expression plasmid after 1 h of treatment with two different concentrations of GOx. The integrity of the genome was monitored by amplification of the 10kb mitochondrial genome-specific DNA fragment and normalized by mitochondrial genome copy number. The graphs are based on PCR reaction of three independently isolated DNA for each experimental point and shown as mean ± standard error (s.e.m.). Myotubes at day 4 of differentiation were used. * indicates p ≤ 0.05. UT, untreated control.

    Techniques Used: Expressing, Western Blot, Transfection, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Isolation

    31) Product Images from "Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation"

    Article Title: Gammaretroviral vector encoding a fluorescent marker to facilitate detection of reprogrammed human fibroblasts during iPSC generation

    Journal: PeerJ

    doi: 10.7717/peerj.224

    Reprogramming factor and pluripotency related mRNA expression in putative iPSC clones. (A) The RF T to RF E ratio, determined by RT-qPCR of total RNA isolated from indicated cell types, is shown for untransduced MRC-5 fibroblasts, vector-transduced MRC-5 fibroblasts (MRC-5 5V) and derived iPSC clones. The RF T /RF E ratios were normalized to that observed in hES cells. Mean ± standard deviation of the combined RF T /RF E ratios of all four RFs are shown above the bars, except for MRC-5 5V where the individual RF T /RF E ratio, while significantly different from hES, also varied significantly between the RFs. (B) Expression of NANOG and DNA methyltransferase 3B ( DNMT3B ) in hES cells, untransduced MRC-5 and transduced MRC-5 (MRC-5 5V), and derived iPSC clones. The mRNA expression was normalized to β-actin levels in the samples. Error bar represents one standard deviation. The standard deviation of the ratio of means was calculated as described under Materials and Methods.
    Figure Legend Snippet: Reprogramming factor and pluripotency related mRNA expression in putative iPSC clones. (A) The RF T to RF E ratio, determined by RT-qPCR of total RNA isolated from indicated cell types, is shown for untransduced MRC-5 fibroblasts, vector-transduced MRC-5 fibroblasts (MRC-5 5V) and derived iPSC clones. The RF T /RF E ratios were normalized to that observed in hES cells. Mean ± standard deviation of the combined RF T /RF E ratios of all four RFs are shown above the bars, except for MRC-5 5V where the individual RF T /RF E ratio, while significantly different from hES, also varied significantly between the RFs. (B) Expression of NANOG and DNA methyltransferase 3B ( DNMT3B ) in hES cells, untransduced MRC-5 and transduced MRC-5 (MRC-5 5V), and derived iPSC clones. The mRNA expression was normalized to β-actin levels in the samples. Error bar represents one standard deviation. The standard deviation of the ratio of means was calculated as described under Materials and Methods.

    Techniques Used: Expressing, Quantitative RT-PCR, Isolation, Plasmid Preparation, Derivative Assay, Clone Assay, Standard Deviation

    Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and
    Figure Legend Snippet: Methylation analysis of Moloney vector 5′ LTR using MSRE-qPCR. (A) Sequence of 5′ long terminal repeat (LTR) and untranslated region of Moloney murine leukemia virus. The U3, R and U5 sequences within the LTR are shown and demarcated by vertical lines. Also shown are direct repeats (DR1 and DR2), Tata box, polyadenylation signal (Poly A), negative control region (NCR), binding site for ELP/NR5A1, and primer binding site (PBS). The CpG nucleotides are marked underneath by ‘*’ to indicate putative sites of methylation. The methylation sensitive SmaI and methylation insensitive MscI restriction enzyme sites are shown in red and green, respectively. > > > and

    Techniques Used: Methylation, Plasmid Preparation, Real-time Polymerase Chain Reaction, Sequencing, Negative Control, Binding Assay

    32) Product Images from "Fidelity Index Determination of DNA Methyltransferases"

    Article Title: Fidelity Index Determination of DNA Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063866

    Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.
    Figure Legend Snippet: Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.

    Techniques Used: Methylation, Activity Assay

    Comparison of a restriction enzyme digestion protection assay and radioactive methylation assay with λ DNA. Both assays were performed using a two-fold dilution series of M.HhaI. The top portion of the figure represents the extent of protection exhibited by M.HhaI against HhaI RE digestion. The bottom portion of the figure shows the amount of H 3 -methyl incorporation by M.HhaI. The X-axis represents the dilution factor of the M.HhaI, where 0 is the highest concentration of enzyme and corresponds to the highest amount of H 3 -methyl incorporation. In contrast, a dilution factor of 19 represents the lowest concentration and enzyme and corresponds to base level H 3 -methyl incorporation. The asterisk designates the LCF, the double dagger designates the HCN, and the plus sign represents the point at which star activity occurs. Upon comparing the same dilution factors from both assays, both can determine the point at which complete methylation of the cognate site occurs. However, there is an apparent increase in H 3 -methyl incorporation after complete methylation of the cognate site at dilution factor 1/2 3 in the radioactive methylation assay, indicating the presence of star activity, but there is no observable difference on the gel at the same dilution factor in the protection assay.
    Figure Legend Snippet: Comparison of a restriction enzyme digestion protection assay and radioactive methylation assay with λ DNA. Both assays were performed using a two-fold dilution series of M.HhaI. The top portion of the figure represents the extent of protection exhibited by M.HhaI against HhaI RE digestion. The bottom portion of the figure shows the amount of H 3 -methyl incorporation by M.HhaI. The X-axis represents the dilution factor of the M.HhaI, where 0 is the highest concentration of enzyme and corresponds to the highest amount of H 3 -methyl incorporation. In contrast, a dilution factor of 19 represents the lowest concentration and enzyme and corresponds to base level H 3 -methyl incorporation. The asterisk designates the LCF, the double dagger designates the HCN, and the plus sign represents the point at which star activity occurs. Upon comparing the same dilution factors from both assays, both can determine the point at which complete methylation of the cognate site occurs. However, there is an apparent increase in H 3 -methyl incorporation after complete methylation of the cognate site at dilution factor 1/2 3 in the radioactive methylation assay, indicating the presence of star activity, but there is no observable difference on the gel at the same dilution factor in the protection assay.

    Techniques Used: Methylation, Concentration Assay, Activity Assay

    33) Product Images from "A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD"

    Article Title: A conserved RpoS-dependent small RNA controls the synthesis of major porin OmpD

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1156

    ( A ) Regulation of OmpD-GFP reporter fusions by SdsR. Salmonella ΔsdsR ΔompD cells carrying the control vector or pP L -SdsR were co-transformed with low-copy plasmids expressing gfp alone or a series of translational ompD::gfp fusions spanning the complete 5′-UTR plus an increasing number of nucleotides of the ompD coding sequence (D+3:: gfp ; D+45:: gfp ; D+78:: gfp ; D+99:: gfp ; see Supplementary Table S2 for details on plasmids) as depicted in (D). Whole-protein samples were collected from cells grown to an OD 600 of 2.0, and regulation of reporter fusions was determined by signal quantification on western blots. Relative GFP levels in the presence of the control plasmid (black bars; set to 100) or the constitutive pP L -SdsR (grey bars); errors indicate standard deviation from three biological replicates. ( B ) Schematic illustration of the 3′-RACE approach employed for target site determination. ( C ) 3′-RACE analysis of ompD mRNA fragments enriched upon SdsR pulse expression. cDNA was prepared from total RNA of Δ sdsR cells as well as the Δ sdsR Δ ompD control strain prior to and at indicated timepoints after SdsR induction from an inducible P BAD promoter. Salmonella genomic DNA (gDNA) served as a control template. DNA fragments were recovered from the indicated band of ∼150 bp (lane 6), and ompD 3′-ends were determined by sequencing of subcloned fragments. ( D ) Location of ompD 3′-ends obtained by 3′-RACE analysis. The ompD :: gfp reporter plasmids and their regulation by SdsR (see Figure 5 A) are represented schematically. The filled circle indicates the approximate coverage of ompD mRNA by the 30S ribosomal subunit binding to the RBS. Position as well as frequency of enriched break-down products determined by 3′-RACE ( Figure 5 C) are shown below the ompD CDS.
    Figure Legend Snippet: ( A ) Regulation of OmpD-GFP reporter fusions by SdsR. Salmonella ΔsdsR ΔompD cells carrying the control vector or pP L -SdsR were co-transformed with low-copy plasmids expressing gfp alone or a series of translational ompD::gfp fusions spanning the complete 5′-UTR plus an increasing number of nucleotides of the ompD coding sequence (D+3:: gfp ; D+45:: gfp ; D+78:: gfp ; D+99:: gfp ; see Supplementary Table S2 for details on plasmids) as depicted in (D). Whole-protein samples were collected from cells grown to an OD 600 of 2.0, and regulation of reporter fusions was determined by signal quantification on western blots. Relative GFP levels in the presence of the control plasmid (black bars; set to 100) or the constitutive pP L -SdsR (grey bars); errors indicate standard deviation from three biological replicates. ( B ) Schematic illustration of the 3′-RACE approach employed for target site determination. ( C ) 3′-RACE analysis of ompD mRNA fragments enriched upon SdsR pulse expression. cDNA was prepared from total RNA of Δ sdsR cells as well as the Δ sdsR Δ ompD control strain prior to and at indicated timepoints after SdsR induction from an inducible P BAD promoter. Salmonella genomic DNA (gDNA) served as a control template. DNA fragments were recovered from the indicated band of ∼150 bp (lane 6), and ompD 3′-ends were determined by sequencing of subcloned fragments. ( D ) Location of ompD 3′-ends obtained by 3′-RACE analysis. The ompD :: gfp reporter plasmids and their regulation by SdsR (see Figure 5 A) are represented schematically. The filled circle indicates the approximate coverage of ompD mRNA by the 30S ribosomal subunit binding to the RBS. Position as well as frequency of enriched break-down products determined by 3′-RACE ( Figure 5 C) are shown below the ompD CDS.

    Techniques Used: Plasmid Preparation, Transformation Assay, Expressing, Sequencing, Western Blot, Standard Deviation, Binding Assay

    34) Product Images from "Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase"

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

    Journal: Oncotarget

    doi:

    Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.
    Figure Legend Snippet: Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Mutagenesis, Sequencing

    The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.
    Figure Legend Snippet: The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.
    Figure Legend Snippet: Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Amplification

    UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.
    Figure Legend Snippet: UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Polymerase Chain Reaction, Amplification

    Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.
    Figure Legend Snippet: Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, In Vitro, Functional Assay, Polymerase Chain Reaction, Amplification, Generated

    The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.
    Figure Legend Snippet: The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.

    Techniques Used: Formalin-fixed Paraffin-Embedded, HRM Assay, Polymerase Chain Reaction, Amplification, Concentration Assay

    35) Product Images from "Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at S?o Paulo city, Brasil"

    Article Title: Oxacilin-resistant Coagulase-negative staphylococci (CoNS) bacteremia in a general hospital at S?o Paulo city, Brasil

    Journal: Brazilian Journal of Microbiology

    doi: 10.1590/S1517-83822008000400006

    PFGE profile of SmaI-digested chromosomal DNA of CoNS isolates, obtained from patients in 9 de Julho Hospital in São Paulo city, Brazil. λ lamba ladder DNA markers; lanes 1–5 S. epidermidis ; lanes 6,7 and 10: S. haemolyticus ; lane 8: S. hominis ; lane 9: S. warneri ; lane 11: S. cohnii spp urealyticus .
    Figure Legend Snippet: PFGE profile of SmaI-digested chromosomal DNA of CoNS isolates, obtained from patients in 9 de Julho Hospital in São Paulo city, Brazil. λ lamba ladder DNA markers; lanes 1–5 S. epidermidis ; lanes 6,7 and 10: S. haemolyticus ; lane 8: S. hominis ; lane 9: S. warneri ; lane 11: S. cohnii spp urealyticus .

    Techniques Used:

    36) Product Images from "Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells"

    Article Title: Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl703

    Effects of induced expression of TFAM-stop and TFAM-MycHis and of RNAi knockdown of TFAM expression. Mitochondrial proteins, DNA and RNA were analysed from Flp-In™ T-Rex™ -293 cells stably transfected with the TFAM-stop (a–c) or TFAM-MycHis construct (d–f), induced over the times indicated or from HEK293T cells (g–i) following transfection with siRNAs Si2 and Si4 over the times indicated. In each case, error bars indicate means ± SEs from at least three independent experiments. a.u., arbitrary units. Measurements of mtDNA levels (a, d and g) are arbitrarily normalized to the mean values for uninduced or untreated cells. For cells under TFAM induction, the measurements were made by two independent methods, Southern blotting and Q-PCR and the values plotted for each time point are the means of measurements by the two methods, shown in Supplementary Figures 1 and 2. ( b , e and h ) show TFAM protein levels normalized to the mtDNA levels shown in ( a , d and g ), then normalized against the level in uninduced or untreated cells. ( c , f and i ) show ND3 mRNA levels normalized first against the 5S rRNA loading control, then against the mtDNA levels shown in (a, d and g), then finally against the level in uninduced or untreated cells. Samples of the raw data and compiled data for TFAM protein, mtDNA and RNA levels on which this figure is based, are shown in Supplementary Figures 1–3.
    Figure Legend Snippet: Effects of induced expression of TFAM-stop and TFAM-MycHis and of RNAi knockdown of TFAM expression. Mitochondrial proteins, DNA and RNA were analysed from Flp-In™ T-Rex™ -293 cells stably transfected with the TFAM-stop (a–c) or TFAM-MycHis construct (d–f), induced over the times indicated or from HEK293T cells (g–i) following transfection with siRNAs Si2 and Si4 over the times indicated. In each case, error bars indicate means ± SEs from at least three independent experiments. a.u., arbitrary units. Measurements of mtDNA levels (a, d and g) are arbitrarily normalized to the mean values for uninduced or untreated cells. For cells under TFAM induction, the measurements were made by two independent methods, Southern blotting and Q-PCR and the values plotted for each time point are the means of measurements by the two methods, shown in Supplementary Figures 1 and 2. ( b , e and h ) show TFAM protein levels normalized to the mtDNA levels shown in ( a , d and g ), then normalized against the level in uninduced or untreated cells. ( c , f and i ) show ND3 mRNA levels normalized first against the 5S rRNA loading control, then against the mtDNA levels shown in (a, d and g), then finally against the level in uninduced or untreated cells. Samples of the raw data and compiled data for TFAM protein, mtDNA and RNA levels on which this figure is based, are shown in Supplementary Figures 1–3.

    Techniques Used: Expressing, Stable Transfection, Transfection, Construct, Southern Blot, Polymerase Chain Reaction

    Effects of TFAM overexpression and knockdown on mtDNA topology. One-dimensional agarose gel blots, hybridized with O H probe. ( a ) mtDNA from uninduced cells and from cells induced to overexpress TFAM-stop for 48 h, fractionated on a 0.4% agarose gel run in TBE. Only the high molecular weight portion of the gel is shown. Samples were either untreated (U) or treated with T7 gp3 endonuclease (gp3), topoisomerase I (tI), topoisomerase IV (tIV) or topoisomerase IV plus T7 gp3 endonuclease. Identity of the main topoisomers was inferred from enzymatic sensitivity and confirmed by other treatments (data not shown). DNA from cells treated with TFAM-specific siRNAs (RNAi) for 24 h was run on a separate gel. ( b ) MtDNA from TFAM-induced, uninduced and siRNA-treated cells, fractionated on 0.4% agarose gels run in TBE. Only the low molecular weight portion of each gel is shown. First 4 lanes of upper panel are equally exposed, whereas the right-most two lanes are ∼3-fold overloaded, to reveal the presence of 7S DNA in induced cells. Samples were either heated for 2 min at 95°C (+) or left unheated, as indicated.
    Figure Legend Snippet: Effects of TFAM overexpression and knockdown on mtDNA topology. One-dimensional agarose gel blots, hybridized with O H probe. ( a ) mtDNA from uninduced cells and from cells induced to overexpress TFAM-stop for 48 h, fractionated on a 0.4% agarose gel run in TBE. Only the high molecular weight portion of the gel is shown. Samples were either untreated (U) or treated with T7 gp3 endonuclease (gp3), topoisomerase I (tI), topoisomerase IV (tIV) or topoisomerase IV plus T7 gp3 endonuclease. Identity of the main topoisomers was inferred from enzymatic sensitivity and confirmed by other treatments (data not shown). DNA from cells treated with TFAM-specific siRNAs (RNAi) for 24 h was run on a separate gel. ( b ) MtDNA from TFAM-induced, uninduced and siRNA-treated cells, fractionated on 0.4% agarose gels run in TBE. Only the low molecular weight portion of each gel is shown. First 4 lanes of upper panel are equally exposed, whereas the right-most two lanes are ∼3-fold overloaded, to reveal the presence of 7S DNA in induced cells. Samples were either heated for 2 min at 95°C (+) or left unheated, as indicated.

    Techniques Used: Over Expression, Agarose Gel Electrophoresis, Molecular Weight

    37) Product Images from "Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay"

    Article Title: Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj522

    L1 insertions from irradiated clones have endonuclease-dependent features. Single L1-EGFP transfected 143B cells expressing EGFP were isolated by flow cytometry and expanded. DNA was extracted and the 3′ genomic flank amplified and sequenced by suppression PCR or inverse PCR (see Materials and Methods). The 5′ flank was then amplified and sequenced using primers designed from the human genome database. Hallmarks of endonuclease-dependent L1 insertion include 7–20 bp target site duplications (TSDs), AT rich consensus target sites and poly-A tails. Dark gray boxes denote TSDs. TSD sequences are displayed beneath each dark gray box. Numbers represent map positions in L1-EGFP; a full length insertion (including the spliced EGFP cassette) is 7814 bp long. Poly-A tail length is given as the subscripted number next to A/T. Chromosome insertion location is given in the 3′ flank. ( A ) L1 insertion flanks from an unirradiated cell. ( B ) Insertion flanks recovered following 4 Gy irradiation resemble most endonuclease-dependent genomic L1 insertions. ( C ) An endonuclease-deficient L1 insertion in a CHO-K1 cell has a deletion at the site of insertion, lacks target site duplications and has 5′ transduced sequence.
    Figure Legend Snippet: L1 insertions from irradiated clones have endonuclease-dependent features. Single L1-EGFP transfected 143B cells expressing EGFP were isolated by flow cytometry and expanded. DNA was extracted and the 3′ genomic flank amplified and sequenced by suppression PCR or inverse PCR (see Materials and Methods). The 5′ flank was then amplified and sequenced using primers designed from the human genome database. Hallmarks of endonuclease-dependent L1 insertion include 7–20 bp target site duplications (TSDs), AT rich consensus target sites and poly-A tails. Dark gray boxes denote TSDs. TSD sequences are displayed beneath each dark gray box. Numbers represent map positions in L1-EGFP; a full length insertion (including the spliced EGFP cassette) is 7814 bp long. Poly-A tail length is given as the subscripted number next to A/T. Chromosome insertion location is given in the 3′ flank. ( A ) L1 insertion flanks from an unirradiated cell. ( B ) Insertion flanks recovered following 4 Gy irradiation resemble most endonuclease-dependent genomic L1 insertions. ( C ) An endonuclease-deficient L1 insertion in a CHO-K1 cell has a deletion at the site of insertion, lacks target site duplications and has 5′ transduced sequence.

    Techniques Used: Irradiation, Clone Assay, Transfection, Expressing, Isolation, Flow Cytometry, Cytometry, Amplification, Polymerase Chain Reaction, Inverse PCR, Sequencing

    38) Product Images from "Generation of FGF reporter transgenic zebrafish and their utility in chemical screens"

    Article Title: Generation of FGF reporter transgenic zebrafish and their utility in chemical screens

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-7-62

    Generation of Dusp6 DNA construct and expression of d2EGFP in transgenic embryos . (A) Diagram showing the Dusp6 gene locus and the DNA construct used in generating transgenic zebrafish. (B, E, H) dusp6 expression at oblong (B) , dome (E) , and shield stage. (C) d2EGFP mRNA expression at sphere stage. (D, F, G, I, J) Tg(Dusp6:d2EGFP) pt 6 embryos at dome (D) , 30% epiboly (F G) , and shield (I J) stage. (B-F I) are lateral views and (H J) are animal views. Red arrowheads mark dorsal region of the embryo.
    Figure Legend Snippet: Generation of Dusp6 DNA construct and expression of d2EGFP in transgenic embryos . (A) Diagram showing the Dusp6 gene locus and the DNA construct used in generating transgenic zebrafish. (B, E, H) dusp6 expression at oblong (B) , dome (E) , and shield stage. (C) d2EGFP mRNA expression at sphere stage. (D, F, G, I, J) Tg(Dusp6:d2EGFP) pt 6 embryos at dome (D) , 30% epiboly (F G) , and shield (I J) stage. (B-F I) are lateral views and (H J) are animal views. Red arrowheads mark dorsal region of the embryo.

    Techniques Used: Construct, Expressing, Transgenic Assay

    39) Product Images from "Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii"

    Article Title: Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030077

    DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.
    Figure Legend Snippet: DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.

    Techniques Used: Sequencing, Isolation, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Southern Blot, Marker, Construct, Transformation Assay

    DNA Replication Origin on the Main Chromosome: oriC1 (A) Sequence features of the ARS element isolated from genomic libraries of H230 DNA, including selected genes (see text for details). Coordinates of plasmid inserts generated by AciI digestion are shown (including insert in pTA313), in addition to the minimal ARS element in pTA441 and pCN11. See Figure 1 A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Above the line are sequences of repeats found at the intergenic region of the H. volcanii chromosomal replication origin (correspond to numbered arrows in Figure 2 A). Below the line are sequences of repeats found at other (presumed) archaeal origins. The species and relevant cdc6/orc1 genes are H. marismortui cdc6–4 (Hmar-1–2), Halobacterium sp. NRC-1 orc7 (NRC1-1-2), Natronomonas pharaonis cdc6–1 (Npha-1–2), S. solfataricus cdc6–1 (Sso-1–2), and P. abyssi cdc6 (Pab-1–4). The orientation is indicated by arrows and conserved positions are shaded. Among halophilic archaea, repeats surrounding the primary DUE feature a longer consensus sequence (Halo-ORB, boxed), which contains the core mini-ORB and “G-string” elements also found in other archaea, plus a halophile-specific “G-string.” (C) Southern blot of PFG of DNA from strain H53, probed with the AciI ARS insert from pTA313.
    Figure Legend Snippet: DNA Replication Origin on the Main Chromosome: oriC1 (A) Sequence features of the ARS element isolated from genomic libraries of H230 DNA, including selected genes (see text for details). Coordinates of plasmid inserts generated by AciI digestion are shown (including insert in pTA313), in addition to the minimal ARS element in pTA441 and pCN11. See Figure 1 A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Above the line are sequences of repeats found at the intergenic region of the H. volcanii chromosomal replication origin (correspond to numbered arrows in Figure 2 A). Below the line are sequences of repeats found at other (presumed) archaeal origins. The species and relevant cdc6/orc1 genes are H. marismortui cdc6–4 (Hmar-1–2), Halobacterium sp. NRC-1 orc7 (NRC1-1-2), Natronomonas pharaonis cdc6–1 (Npha-1–2), S. solfataricus cdc6–1 (Sso-1–2), and P. abyssi cdc6 (Pab-1–4). The orientation is indicated by arrows and conserved positions are shaded. Among halophilic archaea, repeats surrounding the primary DUE feature a longer consensus sequence (Halo-ORB, boxed), which contains the core mini-ORB and “G-string” elements also found in other archaea, plus a halophile-specific “G-string.” (C) Southern blot of PFG of DNA from strain H53, probed with the AciI ARS insert from pTA313.

    Techniques Used: Sequencing, Isolation, Plasmid Preparation, Generated, Southern Blot

    40) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.
    Figure Legend Snippet: rDNA IGS matrix attachment can account for growth- and Myc-dependent changes in higher-order rDNA structure. (A) Distant regions within IGS are bound to matrix in close proximity to each other. The left panel shows the combinations of primer sets used and their locations and orientations along rat rDNA repeat (sequences of primer sets are shown in Supplementary Table S4). The right panels show results for different primer pairs from an MAR-loop assay of growing TGR-1 cells, in which DNA fragments, which are held in close proximity to each other after cleavage with XhoI (X) and BamHI (B) by matrix attachment, can be ligated (+ Lig) to create novel DNA fragments. The panels also show the migration of positive control (C) fragments, which indicate the expected size of potential ligation products, and that no novel DNA fragments are detected in the absence of added ligase (− Lig). The last panel (R0) is a loading control (see Figure 1B ). (B) The MAR-loop assay and 3C assay identify the involvement of an equivalent set of rDNA IGS regions in the formation of rDNA gene loop structures in growing HeLa cells. Annotations are the same as for part (A) (sequences of primer pairs are shown in Supplementary Table S6) except that the H40 region is amplified in all samples as a loading control. (C) Positive proximity results from the MAR-loop assay are predominately associated with the matrix fraction when the assay is performed on isolated matrix-associated (M) and matrix-non-associated (Sup) fractions. Other annotations are shown as (A) and (B). Growth stimulation of matrix-associated gene loop structures is dependent on the activity of c-Myc in (D) HeLa cells and (E) TGR-1 cells. MAR-ligation assay results for starved HeLa and TGR-1 cells before (−serum) or after (+serum) addition of medium containing serum in the absence or presence of c-Myc inhibitor, 10058-F4. Other annotations are as for part (A) and part (B). The filled ramps indicate that PCR amplifications were performed at increasing substrate concentrations, since product formation is easily saturated at higher product concentrations. (F) Myc-ER activation is sufficient to induce matrix-associated gene looping in cells lacking endogenous c-Myc. MAR-ligation assay results before (−4-HT) or after (+4-HT) treatment of Rat1MycER cells. The cutting efficiencies of restriction enzymes on all the indicated samples are shown in Supplementary Figure S2C–E.

    Techniques Used: Migration, Positive Control, Ligation, Amplification, Isolation, Activity Assay, Polymerase Chain Reaction, Activation Assay

    41) Product Images from "Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6"

    Article Title: Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-14-17

    Direct transcriptional regulators of lin-39 in the embryo and larva. A) Horizontal lines represent 20 kb of genomic DNA surrounding the lin-39 locus. The lin-39 transcript is shown below the top line, with boxes representing exons. The next horizontal line shows evolutionarily-conserved regions (ECRs; thin vertical lines), the PCR fragments used in the yeast one hybrid assays containing the ECRs (boxes labeled 1–12), and two fragments ( pJW3.9 shown, JW5 unlabeled) identified previously using an enhancerless GFP assay [ 47 ]. Transcription factors that bind the lin-39 gene are shown above the line (previously reported) or below the line (reported in this work). B) Model for positive feedback loop between egl-18 / elt-6 and lin-39 . EGL-18 and ELT-6 act via the GATA site in enhancer pJW3.9 to facilitate initiation of lin-39 expression in the embryo, and then LIN-39 acts to positively regulate egl-18 / elt-6 expression via the Hox/Pbx binding site in the intron of egl-18 [ 55 ].
    Figure Legend Snippet: Direct transcriptional regulators of lin-39 in the embryo and larva. A) Horizontal lines represent 20 kb of genomic DNA surrounding the lin-39 locus. The lin-39 transcript is shown below the top line, with boxes representing exons. The next horizontal line shows evolutionarily-conserved regions (ECRs; thin vertical lines), the PCR fragments used in the yeast one hybrid assays containing the ECRs (boxes labeled 1–12), and two fragments ( pJW3.9 shown, JW5 unlabeled) identified previously using an enhancerless GFP assay [ 47 ]. Transcription factors that bind the lin-39 gene are shown above the line (previously reported) or below the line (reported in this work). B) Model for positive feedback loop between egl-18 / elt-6 and lin-39 . EGL-18 and ELT-6 act via the GATA site in enhancer pJW3.9 to facilitate initiation of lin-39 expression in the embryo, and then LIN-39 acts to positively regulate egl-18 / elt-6 expression via the Hox/Pbx binding site in the intron of egl-18 [ 55 ].

    Techniques Used: Polymerase Chain Reaction, Labeling, Activated Clotting Time Assay, Expressing, Binding Assay

    42) Product Images from "Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth"

    Article Title: Characterisation of porin genes from Mycobacterium fortuitum and their impact on growth

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-9-31

    Occurrence of porin genes in M. fortuitum . Chromosomal DNA of different strains was digested with SacII and analysed by Southern Blotting using a probe derived from the porM1 sequence. Lane 1: M. fortuitum 10851/03; lane 2: M. fortuitum 10860/03; lane 3: M. fortuitum DSM 46621.
    Figure Legend Snippet: Occurrence of porin genes in M. fortuitum . Chromosomal DNA of different strains was digested with SacII and analysed by Southern Blotting using a probe derived from the porM1 sequence. Lane 1: M. fortuitum 10851/03; lane 2: M. fortuitum 10860/03; lane 3: M. fortuitum DSM 46621.

    Techniques Used: Southern Blot, Derivative Assay, Sequencing

    43) Product Images from "Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi"

    Article Title: Genetic basis for retention of a critical virulence plasmid of Borrelia burgdorferi

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2007.05969.x

    A. PCR analysis of genomic DNA from B. burgdorferi clones transformed with gene inactivation constructs targeting genes in the bbb26–27 region. Template DNAs from transformants are identified below the lanes and PCR amplification targets above the lanes. Template DNA from B31-A illustrates the PCR products from the wild-type alleles of bbb26–27 , bbb26 and bbb27 (lanes 2–5, 15 and 19), whereas template DNAs from the gene inactivation constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ) depict the PCR profiles of the mutated alleles (lanes 6–9, 16 and 20). The PCR products resulting from the clones transformed with the allelic exchange inactivation plasmids are illustrated in lanes 10–13, 17 and 21. The 1 kbp-plus size standards (Invitrogen) were run in lanes 1, 14 and 18 and sizes (base pairs) are indicated to the left of the panel. B. Graphical representation of the bbb26–27 region on cp26 (B31-A) and the cloned pieces of DNA used for the allelic exchange constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ). The 1168 bp region of cp26 between nucleotides 21923 and 23091 was replaced with the 1146 bp flaB p – aadA resistance cassette to create XL-BBB26–27Δ for disruption of both bbb26 and bbb27 . The 742 bp region of cp26 between nucleotides 21923 and 22579 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB26Δ for disruption of bbb26 . The 438 bp region of cp26 between nucleotides 22653 and 23091 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB27Δ for disruption of bbb27 . Locations of the primers used for analysis in (A) are indicated and the sequences are listed in Table S1 .
    Figure Legend Snippet: A. PCR analysis of genomic DNA from B. burgdorferi clones transformed with gene inactivation constructs targeting genes in the bbb26–27 region. Template DNAs from transformants are identified below the lanes and PCR amplification targets above the lanes. Template DNA from B31-A illustrates the PCR products from the wild-type alleles of bbb26–27 , bbb26 and bbb27 (lanes 2–5, 15 and 19), whereas template DNAs from the gene inactivation constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ) depict the PCR profiles of the mutated alleles (lanes 6–9, 16 and 20). The PCR products resulting from the clones transformed with the allelic exchange inactivation plasmids are illustrated in lanes 10–13, 17 and 21. The 1 kbp-plus size standards (Invitrogen) were run in lanes 1, 14 and 18 and sizes (base pairs) are indicated to the left of the panel. B. Graphical representation of the bbb26–27 region on cp26 (B31-A) and the cloned pieces of DNA used for the allelic exchange constructs (XL-BBB26–27Δ, XL-BBB26Δ and XL-BBB27Δ). The 1168 bp region of cp26 between nucleotides 21923 and 23091 was replaced with the 1146 bp flaB p – aadA resistance cassette to create XL-BBB26–27Δ for disruption of both bbb26 and bbb27 . The 742 bp region of cp26 between nucleotides 21923 and 22579 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB26Δ for disruption of bbb26 . The 438 bp region of cp26 between nucleotides 22653 and 23091 was replaced with the 1100 bp flgB p – aacC1 resistance cassette to create XL-BBB27Δ for disruption of bbb27 . Locations of the primers used for analysis in (A) are indicated and the sequences are listed in Table S1 .

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Transformation Assay, Construct, Amplification

    44) Product Images from "Systematic evaluation of genome-wide methylated DNA enrichment using a CpG island array"

    Article Title: Systematic evaluation of genome-wide methylated DNA enrichment using a CpG island array

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-12-10

    MA plots and volcano plots showing data from the linear model fitted to replicate arrays for each of the DMH-v1, DMH-v2, MMASS-v1, MMASS-v2, and MeDIP methods . Colored probes represent external DNA controls, Hex, Alien PCR product, DMSO, empty, and blank, respectively. The results of external DNA controls are nearly consistent with theoretical ratios. Plots of MMASS with either v1 or v2 set of enzymes show higher M, B values and Log 2 fold change than DMH and MeDIP methods.
    Figure Legend Snippet: MA plots and volcano plots showing data from the linear model fitted to replicate arrays for each of the DMH-v1, DMH-v2, MMASS-v1, MMASS-v2, and MeDIP methods . Colored probes represent external DNA controls, Hex, Alien PCR product, DMSO, empty, and blank, respectively. The results of external DNA controls are nearly consistent with theoretical ratios. Plots of MMASS with either v1 or v2 set of enzymes show higher M, B values and Log 2 fold change than DMH and MeDIP methods.

    Techniques Used: Methylated DNA Immunoprecipitation, Polymerase Chain Reaction

    45) Product Images from "Timed Somatic Deletion of p53 in Mice Reveals Age-Associated Differences in Tumor Progression"

    Article Title: Timed Somatic Deletion of p53 in Mice Reveals Age-Associated Differences in Tumor Progression

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006654

    CreERT2-p53 F/F and CreERT2 +/F mice display efficient p53 allele excision in many tissues after tamoxifen treatment. (A) Experimental design to generate mice that can inducibly and somatically delete p53 in many tissues. Floxed p53 alleles are represented by triangle flanked bars. Cre-excised p53 alleles are indicated by solo triangles. (B) Tamoxifen treatment of wildtype mice moderately elevates liver DNA mutation frequency. Three month C57BL/6 Big Blue Mice designed to measure mutation frequencies were treated with tamoxifen (five 1 mg daily injections) and sacrificed at 2 or 4 weeks post-injection and liver DNA subjected to the mutation frequency assay as described in the methods . Mutation frequencies are shown with or without tamoxifen injection (n = 3 for each time point, ±standard error of the mean). (C) Tamoxifen injection of mice has little or no effect on survival compared to vehicle-injected wildtype mice. Tamoxifen injection of CreERT2 negative p53 F/F and p53 +/F mice (red curve, n = 23) showed similar Kaplan-Meier survival curves as wildtype uninjected mice (black curve, n = 55) and vehicle injected CreERT2-p53 +/F mice (blue curve, n = 50). (D) PCR assays show that vehicle-injected CreERT2-p53 +/F tissues exhibit no p53 allele excision, while all tamoxifen-injected CreERT2-p53 +/F tissues show evidence of p53 allele excision. Upper panels show genotyping PCR where the upper band (F) is the larger non-excised floxed allele of p53 and the lower band (+) is the non-floxed wild type p53 allele from various CreERT2-p53 +/F tissues. The lower panels show PCR fragments specific for the excised p53 allele (Δ). The left set of panels contain results from vehicle (corn oil) treated tissues while the right set of panels contain PCR results from tamoxifen treated tissues. Note that in the absence of tamoxifen there is no background p53 allele excision and that in the presence of tamoxifen all tissues show evidence of p53 allele excision. (E) Tamoxifen treatment of CreERT2-p53 +/F and CreERT2-p53 F/F mice results in efficient p53 allele excision (Δ) in some, but not all tissues. Southern blot analysis of genomic DNA from various CreERT2-p53 +/F and CreERT2-p53 F/F tissues was performed as described in the methods . Note that spleen and liver show efficient excision, lung and kidney show partial excision, while eye and brain show little evidence of excision.
    Figure Legend Snippet: CreERT2-p53 F/F and CreERT2 +/F mice display efficient p53 allele excision in many tissues after tamoxifen treatment. (A) Experimental design to generate mice that can inducibly and somatically delete p53 in many tissues. Floxed p53 alleles are represented by triangle flanked bars. Cre-excised p53 alleles are indicated by solo triangles. (B) Tamoxifen treatment of wildtype mice moderately elevates liver DNA mutation frequency. Three month C57BL/6 Big Blue Mice designed to measure mutation frequencies were treated with tamoxifen (five 1 mg daily injections) and sacrificed at 2 or 4 weeks post-injection and liver DNA subjected to the mutation frequency assay as described in the methods . Mutation frequencies are shown with or without tamoxifen injection (n = 3 for each time point, ±standard error of the mean). (C) Tamoxifen injection of mice has little or no effect on survival compared to vehicle-injected wildtype mice. Tamoxifen injection of CreERT2 negative p53 F/F and p53 +/F mice (red curve, n = 23) showed similar Kaplan-Meier survival curves as wildtype uninjected mice (black curve, n = 55) and vehicle injected CreERT2-p53 +/F mice (blue curve, n = 50). (D) PCR assays show that vehicle-injected CreERT2-p53 +/F tissues exhibit no p53 allele excision, while all tamoxifen-injected CreERT2-p53 +/F tissues show evidence of p53 allele excision. Upper panels show genotyping PCR where the upper band (F) is the larger non-excised floxed allele of p53 and the lower band (+) is the non-floxed wild type p53 allele from various CreERT2-p53 +/F tissues. The lower panels show PCR fragments specific for the excised p53 allele (Δ). The left set of panels contain results from vehicle (corn oil) treated tissues while the right set of panels contain PCR results from tamoxifen treated tissues. Note that in the absence of tamoxifen there is no background p53 allele excision and that in the presence of tamoxifen all tissues show evidence of p53 allele excision. (E) Tamoxifen treatment of CreERT2-p53 +/F and CreERT2-p53 F/F mice results in efficient p53 allele excision (Δ) in some, but not all tissues. Southern blot analysis of genomic DNA from various CreERT2-p53 +/F and CreERT2-p53 F/F tissues was performed as described in the methods . Note that spleen and liver show efficient excision, lung and kidney show partial excision, while eye and brain show little evidence of excision.

    Techniques Used: Mouse Assay, Mutagenesis, Injection, Polymerase Chain Reaction, Southern Blot

    46) Product Images from "Producing Proficient Methyl Donors from Alternative Substrates of S-Adenosylmethionine Synthetase"

    Article Title: Producing Proficient Methyl Donors from Alternative Substrates of S-Adenosylmethionine Synthetase

    Journal: Biochemistry

    doi: 10.1021/bi401556p

    DNA methylation by M.SssI using different AdoMet analogues produced by the AdoMet synthetase-catalyzed activation of methionine derivatives. The methionine substrates included l -methionine (Met), l -methionine methyl (MME) and ethyl (MEE) esters produced by C. jejuni AdoMet synthetase and tested at 100 ± 5 μM, and N -acetyl- l -methionine (NAM) and N , N -dimethylmethionine (DMM) produced by N. meningitidis AdoMet synthetase and tested at ∼10 and
    Figure Legend Snippet: DNA methylation by M.SssI using different AdoMet analogues produced by the AdoMet synthetase-catalyzed activation of methionine derivatives. The methionine substrates included l -methionine (Met), l -methionine methyl (MME) and ethyl (MEE) esters produced by C. jejuni AdoMet synthetase and tested at 100 ± 5 μM, and N -acetyl- l -methionine (NAM) and N , N -dimethylmethionine (DMM) produced by N. meningitidis AdoMet synthetase and tested at ∼10 and

    Techniques Used: DNA Methylation Assay, Produced, Activation Assay

    Optimization of AdoMet production catalyzed by AdoMet synthetase. The products of a coupled reaction are shown, where AdoMet synthetase produces AdoMet, which is then used by M.SssI to methylate substrate DNA (linearized pUC19). The concentrations of substrates l -methionine and MgATP were each increased from 0.5 to 6 mM for the 60 min reaction time, followed by restriction digestion with HpaII and gel electrophoresis, shown as a negative image of an ethidium-stained agarose gel, to determine the extent of DNA methylation achieved with each substrate level.
    Figure Legend Snippet: Optimization of AdoMet production catalyzed by AdoMet synthetase. The products of a coupled reaction are shown, where AdoMet synthetase produces AdoMet, which is then used by M.SssI to methylate substrate DNA (linearized pUC19). The concentrations of substrates l -methionine and MgATP were each increased from 0.5 to 6 mM for the 60 min reaction time, followed by restriction digestion with HpaII and gel electrophoresis, shown as a negative image of an ethidium-stained agarose gel, to determine the extent of DNA methylation achieved with each substrate level.

    Techniques Used: Nucleic Acid Electrophoresis, Staining, Agarose Gel Electrophoresis, DNA Methylation Assay

    Restriction endonuclease analysis of pUC19 DNA after methylation. DNA was incubated for 30 or 60 min with either 160 or 320 μM AdoMet at a fixed amount of M.SssI DNA methyltransferase in the presence or absence of added MgCl 2 , followed by restriction digestion with HpaII.
    Figure Legend Snippet: Restriction endonuclease analysis of pUC19 DNA after methylation. DNA was incubated for 30 or 60 min with either 160 or 320 μM AdoMet at a fixed amount of M.SssI DNA methyltransferase in the presence or absence of added MgCl 2 , followed by restriction digestion with HpaII.

    Techniques Used: Methylation, Incubation

    47) Product Images from "Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains"

    Article Title: Whole genome bisulfite sequencing of cell-free DNA and its cellular contributors uncovers placenta hypomethylated domains

    Journal: Genome Biology

    doi: 10.1186/s13059-015-0645-x

    Methylome of ccf DNA isolated from pregnant plasma. (a) Cytosine methylation in non-pregnant and pregnant ccf DNA for CpG, CHG, and CHH contexts are shown. P values were calculated using a Wilcox rank sum test. (b) Methylation of all cytosines located within the DMRs hypermethylated in placenta tissue relative to non-pregnant ccf DNA. The y-axis (density) is the defined as the proportion of CpG sites at a given methylation level. (c) Methylation of all cytosines located within the DMRs hypermethylated in non-pregnant ccf DNA relative to placenta tissue. The y-axis (density) is the defined as the proportion of CpG sites at a given methylation level.
    Figure Legend Snippet: Methylome of ccf DNA isolated from pregnant plasma. (a) Cytosine methylation in non-pregnant and pregnant ccf DNA for CpG, CHG, and CHH contexts are shown. P values were calculated using a Wilcox rank sum test. (b) Methylation of all cytosines located within the DMRs hypermethylated in placenta tissue relative to non-pregnant ccf DNA. The y-axis (density) is the defined as the proportion of CpG sites at a given methylation level. (c) Methylation of all cytosines located within the DMRs hypermethylated in non-pregnant ccf DNA relative to placenta tissue. The y-axis (density) is the defined as the proportion of CpG sites at a given methylation level.

    Techniques Used: Isolation, Methylation

    Linkage between fragment size and local DNA methylation in non-pregnant ccf DNA. (a) Fragment size of ccf DNA as measured by WGBS. Each line represents an individual ccf sample. Loss of representation at approximately 92 to 98 bp is an artifact of adapter trimming prior to alignment. (b) Ratio of methylated CpG, CHG, and CHH cytosines within large fragments ( > 200 bp) relative to methylated cytosines in small fragments (
    Figure Legend Snippet: Linkage between fragment size and local DNA methylation in non-pregnant ccf DNA. (a) Fragment size of ccf DNA as measured by WGBS. Each line represents an individual ccf sample. Loss of representation at approximately 92 to 98 bp is an artifact of adapter trimming prior to alignment. (b) Ratio of methylated CpG, CHG, and CHH cytosines within large fragments ( > 200 bp) relative to methylated cytosines in small fragments (

    Techniques Used: DNA Methylation Assay, Methylation

    Methylation patterns in buffy coat, placenta, and non-pregnant ccf DNA. (a) The distribution of mean CpG methylation for each sample type (non-pregnant ccf DNA, maternal buffy coat, and placenta). The y-axis represents the relative proportion of all evaluated CpG dinucleotides exhibiting a particular level of CpG methylation. The histogram bins each have a width of 1%. (b) CpG methylation of non-pregnant ccf DNA samples was assessed in ENCODE-defined enriched regions for H3K4me1, H3K4me3, H3K9me3, and H3K27me3. Unenriched data were generated by a random sampling of the same number of CpG sites as used for enrichment, but located elsewhere in the genome. The width of each violin plot is representative of data density at a given CpG methylation level. (c) The number of DMRs more methylated in placenta (red) and non-pregnant (NP) ccf DNA (blue).
    Figure Legend Snippet: Methylation patterns in buffy coat, placenta, and non-pregnant ccf DNA. (a) The distribution of mean CpG methylation for each sample type (non-pregnant ccf DNA, maternal buffy coat, and placenta). The y-axis represents the relative proportion of all evaluated CpG dinucleotides exhibiting a particular level of CpG methylation. The histogram bins each have a width of 1%. (b) CpG methylation of non-pregnant ccf DNA samples was assessed in ENCODE-defined enriched regions for H3K4me1, H3K4me3, H3K9me3, and H3K27me3. Unenriched data were generated by a random sampling of the same number of CpG sites as used for enrichment, but located elsewhere in the genome. The width of each violin plot is representative of data density at a given CpG methylation level. (c) The number of DMRs more methylated in placenta (red) and non-pregnant (NP) ccf DNA (blue).

    Techniques Used: Methylation, CpG Methylation Assay, Generated, Sampling

    Identification of placenta hypomethylated domains (PHDs). (a) Mean methylation per 50 kbp genomic bin on chromosome 16 with non-pregnant ccf DNA (NP ccf DNA) and placenta shown. CpG sites (blue) and genes (orange) were summed per 50kbp genomic bin. (b) Genomic methylation level by CpG density at 50 kbp bin level. Values on the x-axis represent the number of CpG sites per 50 kbp bin. Numbers along the top indicate the number of genomic bins analyzed. (c) Differential methylation between placenta and non-pregnant plasma as a function of CpG density at 50 kbp bin level. A negative value on the y-axis is indicative of placenta hypomethylation. The red line corresponds to a loess smoothed fit.
    Figure Legend Snippet: Identification of placenta hypomethylated domains (PHDs). (a) Mean methylation per 50 kbp genomic bin on chromosome 16 with non-pregnant ccf DNA (NP ccf DNA) and placenta shown. CpG sites (blue) and genes (orange) were summed per 50kbp genomic bin. (b) Genomic methylation level by CpG density at 50 kbp bin level. Values on the x-axis represent the number of CpG sites per 50 kbp bin. Numbers along the top indicate the number of genomic bins analyzed. (c) Differential methylation between placenta and non-pregnant plasma as a function of CpG density at 50 kbp bin level. A negative value on the y-axis is indicative of placenta hypomethylation. The red line corresponds to a loess smoothed fit.

    Techniques Used: Methylation

    48) Product Images from "Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.)"

    Article Title: Barley Stripe Mosaic Virus (BSMV) Induced MicroRNA Silencing in Common Wheat (Triticum aestivum L.)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126621

    Schematic diagram of integrating BSMV-γb vector and AtIPS1 -based MIM or STTM sequences. Modified BSMV-γb vector (pCaBS-γ-LIC) was shown in this figure. AtIPS1 -based MIM or STTM sequences can be cloned into pCaBS-γ-LIC derivatives by the LIC reaction. MIM structure contained an AtIPS1 backbone, but the target mimic motif of AthmiR399 was changed to that of corresponding miRNAs. STTM structure contained two tandem target mimics separated by a 48 nt imperfect stem-loop linker as described [ 16 ].
    Figure Legend Snippet: Schematic diagram of integrating BSMV-γb vector and AtIPS1 -based MIM or STTM sequences. Modified BSMV-γb vector (pCaBS-γ-LIC) was shown in this figure. AtIPS1 -based MIM or STTM sequences can be cloned into pCaBS-γ-LIC derivatives by the LIC reaction. MIM structure contained an AtIPS1 backbone, but the target mimic motif of AthmiR399 was changed to that of corresponding miRNAs. STTM structure contained two tandem target mimics separated by a 48 nt imperfect stem-loop linker as described [ 16 ].

    Techniques Used: Plasmid Preparation, Modification, Clone Assay

    49) Product Images from "Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis"

    Article Title: Identification of Rv3852 as an Agrimophol-Binding Protein in Mycobacterium tuberculosis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0126211

    Construction of rv3852 knockout Mtb and verification by Southern blot and PCR. (A) Upper panel displays the genetic organization of the rv3852 region in Mtb (WT), Lower panel displays the same region with replacement of rv3852 by hygromycin resistance gene in rv3852 knockout Mtb (KO). Filled rectangle indicates the location of probe used in the Southern blot. Sites of digestion by BclI on genomic DNA from WT and KO as well as the sizes of the generated DNA fragment (a and b) are demonstrated under each panel. PCR products from genomic DNA from WT (1 and 2) and KO (3 and 4) are denoted. (B) Left, Southern blot of BclI digested genomic DNA from WT and KO. Calculated sizes of the fragments hybridizing with the probe were 3714 bp (WT) and 2916 bp (KO) as indicated in A. Right, PCR products from genomic DNA from WT and KO. The calculated sizes of the PCR products were 902 (Lane 1), 860 (Lane 2) for WT and 1322 (Lane 3), 1560 bp (Lane 4) for KO as indicated in A.
    Figure Legend Snippet: Construction of rv3852 knockout Mtb and verification by Southern blot and PCR. (A) Upper panel displays the genetic organization of the rv3852 region in Mtb (WT), Lower panel displays the same region with replacement of rv3852 by hygromycin resistance gene in rv3852 knockout Mtb (KO). Filled rectangle indicates the location of probe used in the Southern blot. Sites of digestion by BclI on genomic DNA from WT and KO as well as the sizes of the generated DNA fragment (a and b) are demonstrated under each panel. PCR products from genomic DNA from WT (1 and 2) and KO (3 and 4) are denoted. (B) Left, Southern blot of BclI digested genomic DNA from WT and KO. Calculated sizes of the fragments hybridizing with the probe were 3714 bp (WT) and 2916 bp (KO) as indicated in A. Right, PCR products from genomic DNA from WT and KO. The calculated sizes of the PCR products were 902 (Lane 1), 860 (Lane 2) for WT and 1322 (Lane 3), 1560 bp (Lane 4) for KO as indicated in A.

    Techniques Used: Knock-Out, Southern Blot, Polymerase Chain Reaction, Generated

    50) Product Images from "Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons"

    Article Title: Rapid Quantification of Mutant Fitness in Diverse Bacteria by Sequencing Randomly Bar-Coded Transposons

    Journal: mBio

    doi: 10.1128/mBio.00306-15

    Comparison of RB-TnSeq to other technologies. (A) Comparison of gene fitness for P. stutzeri grown in a defined medium with glucose as determined with BarSeq ( x axis) or sequencing the transposon-genome insertion junctions (TnSeq; y axis), starting from the same samples of genomic DNA. Genes marked in green have statistically significant phenotypes as determined by BarSeq. The dashed black line marks x = y . (B) Same as panel A for S. amazonensis grown in a defined medium with d , l -lactate. (C) Comparison of S. oneidensis gene fitness in defined medium with l -lactate calculated from BarSeq ( x axis) and previously described data that used mutant libraries with defined DNA bar codes and microarrays to assay strain abundance ( y axis) ( 2 ). The dashed black line marks x = y . (D) BarSeq fitness data for E. coli genes grown in acetate ( x axis) or glucosamine ( y axis) as the sole source of carbon. Genes marked in red have an acetate-specific fitness defect while those marked in blue have a glucosamine-specific fitness defect in the Nichols et al. data set, with thresholds of S
    Figure Legend Snippet: Comparison of RB-TnSeq to other technologies. (A) Comparison of gene fitness for P. stutzeri grown in a defined medium with glucose as determined with BarSeq ( x axis) or sequencing the transposon-genome insertion junctions (TnSeq; y axis), starting from the same samples of genomic DNA. Genes marked in green have statistically significant phenotypes as determined by BarSeq. The dashed black line marks x = y . (B) Same as panel A for S. amazonensis grown in a defined medium with d , l -lactate. (C) Comparison of S. oneidensis gene fitness in defined medium with l -lactate calculated from BarSeq ( x axis) and previously described data that used mutant libraries with defined DNA bar codes and microarrays to assay strain abundance ( y axis) ( 2 ). The dashed black line marks x = y . (D) BarSeq fitness data for E. coli genes grown in acetate ( x axis) or glucosamine ( y axis) as the sole source of carbon. Genes marked in red have an acetate-specific fitness defect while those marked in blue have a glucosamine-specific fitness defect in the Nichols et al. data set, with thresholds of S

    Techniques Used: Sequencing, Mutagenesis

    51) Product Images from "Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation"

    Article Title: Reconstitution of a eukaryotic replisome reveals suppression mechanisms that define leading/lagging strand operation

    Journal: eLife

    doi: 10.7554/eLife.04988

    Pol α requires CMG for priming activity during unwinding of forked DNA. ( A ) Scheme of assays comparing Pol α activity using either CMG helicase or the strand displacing ϕ29 polymerase. ( B ) Autoradiograph of DNA products using either 32 P-dCTP (leading) or 32 P-dGTP (lagging). Use of a DNA-primed leading strand fork (PF) or an unprimed fork (UF) is indicated in the figure. Pol α was present at 10 nM, and reactions were for 20 min. Lanes 1 and 2 represent control reactions of ϕ29 polymerase alone. DOI: http://dx.doi.org/10.7554/eLife.04988.006
    Figure Legend Snippet: Pol α requires CMG for priming activity during unwinding of forked DNA. ( A ) Scheme of assays comparing Pol α activity using either CMG helicase or the strand displacing ϕ29 polymerase. ( B ) Autoradiograph of DNA products using either 32 P-dCTP (leading) or 32 P-dGTP (lagging). Use of a DNA-primed leading strand fork (PF) or an unprimed fork (UF) is indicated in the figure. Pol α was present at 10 nM, and reactions were for 20 min. Lanes 1 and 2 represent control reactions of ϕ29 polymerase alone. DOI: http://dx.doi.org/10.7554/eLife.04988.006

    Techniques Used: Activity Assay, Autoradiography

    Okazaki Fragments are produced along the entire DNA. ( A ) Restriction enzyme map of the 3.2 kb substrate for Psi I and Ear I. ( B ) Lagging strand reactions were performed as detailed in ‘Materials and methods’ using an unprimed forked DNA, CMG, RPA, and either Pol α (lanes 4–6) or Pol α and Pol ε (lanes 7–9), then were either untreated (lanes 4, 7), treated with Psi I (lanes 5, 8), or treated with Ear I (lanes 6, 9). A control leading strand reaction using only ϕ29 Pol is shown in lanes 10–12. Pol α without CMG (lanes 1–3) and ϕ29 alone (lanes 13–15) gave no lagging strand products. The (*) mark incomplete digestion products. The reaction products were analyzed on a native 2% agarose gel. DOI: http://dx.doi.org/10.7554/eLife.04988.007
    Figure Legend Snippet: Okazaki Fragments are produced along the entire DNA. ( A ) Restriction enzyme map of the 3.2 kb substrate for Psi I and Ear I. ( B ) Lagging strand reactions were performed as detailed in ‘Materials and methods’ using an unprimed forked DNA, CMG, RPA, and either Pol α (lanes 4–6) or Pol α and Pol ε (lanes 7–9), then were either untreated (lanes 4, 7), treated with Psi I (lanes 5, 8), or treated with Ear I (lanes 6, 9). A control leading strand reaction using only ϕ29 Pol is shown in lanes 10–12. Pol α without CMG (lanes 1–3) and ϕ29 alone (lanes 13–15) gave no lagging strand products. The (*) mark incomplete digestion products. The reaction products were analyzed on a native 2% agarose gel. DOI: http://dx.doi.org/10.7554/eLife.04988.007

    Techniques Used: Produced, Recombinase Polymerase Amplification, Agarose Gel Electrophoresis

    52) Product Images from "A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs"

    Article Title: A structural determinant in the uracil DNA glycosylase superfamily for the removal of uracil from adenine/uracil base pairs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1332

    Substrates, sequence alignment, structure and UDG activity. ( A ) Sequences of uracil-containing DNA substrates. ( B ) Sequence alignment of E. coli MUG (GenBank accession number P0A9H1.1), E. coli UNG (GenBank accession number NP_417075.1) and Tth UDGb (GenBank accession number YP_144415.1). ( C ) Structure of E. coli MUG (PDB 1MUG) with uracil. Motifs 1 and 2 are shown in orange and purple, respectively. K68 and uracil are colored by atom type. ( D ) DNA glycosylase activity of MUG-WT on uracil-containing substrates. Cleavage reactions were performed as described in the Materials and Methods section with 100 nM MUG-WT protein and 10 nM substrate. ( E ) DNA glycosylase activity of MUG-K68N on uracil-containing substrates. Cleavage reactions were performed as described in the Materials and Methods section with 100 nM MUG-K68N protein and 10 nM substrate.
    Figure Legend Snippet: Substrates, sequence alignment, structure and UDG activity. ( A ) Sequences of uracil-containing DNA substrates. ( B ) Sequence alignment of E. coli MUG (GenBank accession number P0A9H1.1), E. coli UNG (GenBank accession number NP_417075.1) and Tth UDGb (GenBank accession number YP_144415.1). ( C ) Structure of E. coli MUG (PDB 1MUG) with uracil. Motifs 1 and 2 are shown in orange and purple, respectively. K68 and uracil are colored by atom type. ( D ) DNA glycosylase activity of MUG-WT on uracil-containing substrates. Cleavage reactions were performed as described in the Materials and Methods section with 100 nM MUG-WT protein and 10 nM substrate. ( E ) DNA glycosylase activity of MUG-K68N on uracil-containing substrates. Cleavage reactions were performed as described in the Materials and Methods section with 100 nM MUG-K68N protein and 10 nM substrate.

    Techniques Used: Sequencing, Activity Assay

    53) Product Images from "Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium"

    Article Title: Systematic mapping of two component response regulators to gene targets in a model sulfate reducing bacterium

    Journal: Genome Biology

    doi: 10.1186/gb-2011-12-10-r99

    The DNA-affinity-purified-chip (DAP-chip) method . (a) DAP-chip strategy. The D. vulgaris RR gene is cloned into E. coli with a carboxy-terminal His-tag. Purified His-tagged protein is phosphorylated with acetyl phosphate, and mixed with sheared D. vulgaris genomic DNA. An aliquot of the binding reaction is saved as input DNA, while the rest is subjected to affinity purification using Ni-NTA resin. The input and the RR-bound DNA are whole genome amplified, and labeled with Cy3 and Cy5, respectively. The labeled DNA is pooled together and hybridized to a tiling array, which is then analyzed to determine the gene targets. (b) Summary of DAP-chip workflow. The flowchart shows a summary of results at the following steps: protein purification, positive target determination, quantitative PCR (qPCR) test for target enrichment, DAP-chip hybridization, target list determination, and binding site motif validation. AAA, σ54 interaction domain; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; NTA, nitrilotriacetic acid; REC, receiver domain; RR: response regulator.
    Figure Legend Snippet: The DNA-affinity-purified-chip (DAP-chip) method . (a) DAP-chip strategy. The D. vulgaris RR gene is cloned into E. coli with a carboxy-terminal His-tag. Purified His-tagged protein is phosphorylated with acetyl phosphate, and mixed with sheared D. vulgaris genomic DNA. An aliquot of the binding reaction is saved as input DNA, while the rest is subjected to affinity purification using Ni-NTA resin. The input and the RR-bound DNA are whole genome amplified, and labeled with Cy3 and Cy5, respectively. The labeled DNA is pooled together and hybridized to a tiling array, which is then analyzed to determine the gene targets. (b) Summary of DAP-chip workflow. The flowchart shows a summary of results at the following steps: protein purification, positive target determination, quantitative PCR (qPCR) test for target enrichment, DAP-chip hybridization, target list determination, and binding site motif validation. AAA, σ54 interaction domain; DBD, DNA binding domain; EMSA, electrophoretic mobility shift assay; NTA, nitrilotriacetic acid; REC, receiver domain; RR: response regulator.

    Techniques Used: Affinity Purification, Chromatin Immunoprecipitation, Clone Assay, Purification, Binding Assay, Amplification, Labeling, Protein Purification, Real-time Polymerase Chain Reaction, Hybridization, Electrophoretic Mobility Shift Assay

    54) Product Images from "Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania"

    Article Title: Apoptosis-like programmed cell death induces antisense ribosomal RNA (rRNA) fragmentation and rRNA degradation in Leishmania

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2012.85

    The ATP-dependent DEAD-box RNA helicase HEL67 interacts with both the sLSU- γ and asLSU- γ rRNAs to prevent asrRNA fragmentation. ( a ) A modified UV-crosslinking method was used to identify protein factors bound to in vitro -transcribed sLSU- γ and asLSU- γ rRNAs. The 67 kDa and 30 kDa proteins bound to both sLSU- γ and asLSU- γ rRNAs are indicated. ( b , left panel) Strategy to generate a L. infantum null mutant strain ( Lin HEL (−/−) ) for the LinJ.32.0410 gene encoding an ATP-dependent RNA helicase of 67 kDa (HEL67). Both alleles of the HEL67 gene were replaced by the hygromycin phosphotransferase gene ( HYG ) and neomycin phosphotransferase gene ( NEO ) cassettes, respectively, through homologous recombination. An add-back mutant ( Lin HEL (−/−) REV, b , bottom panel) was generated by overexpressing HEL67 as part of the pSP α ZEO α -HEL67 vector in the Lin HEL67 (−/−) mutant background. ( b , right panel) Southern blot hybridization of genomic DNA digested with Xba I and Blp I using the HEL67 3‘-flank sequence as a probe. In Lin HEL67 (−/−) , two hybridizing bands of 2.3 kb (for the HYG gene replacement) and 2.1 kb (for the NEO gene replacement) were detected but not the 3.1 kb HEL67 endogenous band. ( c ) Primer extension analysis was performed to detect asLSU- γ RNA fragmentation using the end-labeled forward primer corresponding to nucleotides 101–118 of the LSU- γ rRNA. ( c , left panel) RNA was extracted from wild-type (WT), Lin HEL67 (−/−) and Lin HEL67 (−/−) REV L. infantum promastigotes subjected to O/N temperature (37 °C) and pH (5.5) stress. MF (15 μ M)-treated L. infantum axenic amastigotes were used as a positive control for the induction of apoptosis and asLSU- γ RNA fragmentation. ( c , right panel) SS-qRT-PCR to detect asLSU- γ RNA levels in WT, Lin HEL67 (−/−) and Lin HEL67 (−/−) REV. A primer corresponding to nucleotides 1–18 of sLSU- γ rRNA was used for cDNA synthesis. ( d ) Primer extension analysis using a reverse primer complementary to nucleotides 196–213 of the sLSU- γ rRNA
    Figure Legend Snippet: The ATP-dependent DEAD-box RNA helicase HEL67 interacts with both the sLSU- γ and asLSU- γ rRNAs to prevent asrRNA fragmentation. ( a ) A modified UV-crosslinking method was used to identify protein factors bound to in vitro -transcribed sLSU- γ and asLSU- γ rRNAs. The 67 kDa and 30 kDa proteins bound to both sLSU- γ and asLSU- γ rRNAs are indicated. ( b , left panel) Strategy to generate a L. infantum null mutant strain ( Lin HEL (−/−) ) for the LinJ.32.0410 gene encoding an ATP-dependent RNA helicase of 67 kDa (HEL67). Both alleles of the HEL67 gene were replaced by the hygromycin phosphotransferase gene ( HYG ) and neomycin phosphotransferase gene ( NEO ) cassettes, respectively, through homologous recombination. An add-back mutant ( Lin HEL (−/−) REV, b , bottom panel) was generated by overexpressing HEL67 as part of the pSP α ZEO α -HEL67 vector in the Lin HEL67 (−/−) mutant background. ( b , right panel) Southern blot hybridization of genomic DNA digested with Xba I and Blp I using the HEL67 3‘-flank sequence as a probe. In Lin HEL67 (−/−) , two hybridizing bands of 2.3 kb (for the HYG gene replacement) and 2.1 kb (for the NEO gene replacement) were detected but not the 3.1 kb HEL67 endogenous band. ( c ) Primer extension analysis was performed to detect asLSU- γ RNA fragmentation using the end-labeled forward primer corresponding to nucleotides 101–118 of the LSU- γ rRNA. ( c , left panel) RNA was extracted from wild-type (WT), Lin HEL67 (−/−) and Lin HEL67 (−/−) REV L. infantum promastigotes subjected to O/N temperature (37 °C) and pH (5.5) stress. MF (15 μ M)-treated L. infantum axenic amastigotes were used as a positive control for the induction of apoptosis and asLSU- γ RNA fragmentation. ( c , right panel) SS-qRT-PCR to detect asLSU- γ RNA levels in WT, Lin HEL67 (−/−) and Lin HEL67 (−/−) REV. A primer corresponding to nucleotides 1–18 of sLSU- γ rRNA was used for cDNA synthesis. ( d ) Primer extension analysis using a reverse primer complementary to nucleotides 196–213 of the sLSU- γ rRNA

    Techniques Used: Modification, In Vitro, Mutagenesis, Homologous Recombination, Generated, Plasmid Preparation, Southern Blot, Hybridization, Sequencing, Labeling, Positive Control, Quantitative RT-PCR

    Overexpression of asLSU- γ rRNA stimulates srRNA degradation upon induction of apoptosis. ( a ) Schematic diagram of Leishmania expression vectors harboring the full-length LSU- γ (213 bp) and part of the LSU- α and LSU- β in the sense (s) and antisense (as) orientation. ( b ) qRT-PCR to validate overexpression of the asLSU- γ RNA in the asLSU1.2 overexpressor in comparison with the sLSU1.2 overexpressor. ( c , upper panel) EtBr-stained RNA gel of MF-treated parasites overexpressing either the sLSU1.2 (0–20 μ M) or the asLSU1.2 (0–15 μ M) rRNA. ( c , bottom panel) RNA blot with the 173 nt ss-DNA probe recognizing asLSU- γ RNA showing more accumulation of the mature asLSU- γ RNA in the untreated asLSU1.2 overexpressor but increased degradation of this RNA upon MF treatment. ( d ) Primer extension analysis to detect sLSU- γ rRNA and its degradation products in both sLSU1.2- and asLSU1.2-overexpressed strains using a reverse primer complementary to nucleotides 196–213 of sLSU- γ
    Figure Legend Snippet: Overexpression of asLSU- γ rRNA stimulates srRNA degradation upon induction of apoptosis. ( a ) Schematic diagram of Leishmania expression vectors harboring the full-length LSU- γ (213 bp) and part of the LSU- α and LSU- β in the sense (s) and antisense (as) orientation. ( b ) qRT-PCR to validate overexpression of the asLSU- γ RNA in the asLSU1.2 overexpressor in comparison with the sLSU1.2 overexpressor. ( c , upper panel) EtBr-stained RNA gel of MF-treated parasites overexpressing either the sLSU1.2 (0–20 μ M) or the asLSU1.2 (0–15 μ M) rRNA. ( c , bottom panel) RNA blot with the 173 nt ss-DNA probe recognizing asLSU- γ RNA showing more accumulation of the mature asLSU- γ RNA in the untreated asLSU1.2 overexpressor but increased degradation of this RNA upon MF treatment. ( d ) Primer extension analysis to detect sLSU- γ rRNA and its degradation products in both sLSU1.2- and asLSU1.2-overexpressed strains using a reverse primer complementary to nucleotides 196–213 of sLSU- γ

    Techniques Used: Over Expression, Expressing, Quantitative RT-PCR, Staining, Northern blot

    55) Product Images from "Genetics, structure, and prevalence of FP967 (CDC Triffid) T-DNA in flax"

    Article Title: Genetics, structure, and prevalence of FP967 (CDC Triffid) T-DNA in flax

    Journal: SpringerPlus

    doi: 10.1186/s40064-015-0923-9

    Sequence adjacent to the LB region in FP967 and SAT2-LB event specific assay. Our analysis indicated a region of the T-DNA between the NOS promoter of NPTII and SAT2 was duplicated between the FFS1 region of the flax genome and the LB of the T-DNA. A qPCR assay was developed to detect this event specific fragment (P85, P86 and prb28). The sequence of these primers and probe is indicated with green and red blocks, respectively. The location of the Spectinomycin resistance gene AADA and SAT2 are shown in yellow and the LB region in grey. The sequence of this fragment was confirmed by PCR sequencing of P30-P85 and P30-P20 fragments.
    Figure Legend Snippet: Sequence adjacent to the LB region in FP967 and SAT2-LB event specific assay. Our analysis indicated a region of the T-DNA between the NOS promoter of NPTII and SAT2 was duplicated between the FFS1 region of the flax genome and the LB of the T-DNA. A qPCR assay was developed to detect this event specific fragment (P85, P86 and prb28). The sequence of these primers and probe is indicated with green and red blocks, respectively. The location of the Spectinomycin resistance gene AADA and SAT2 are shown in yellow and the LB region in grey. The sequence of this fragment was confirmed by PCR sequencing of P30-P85 and P30-P20 fragments.

    Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Cartoon of the insertion of the FP967 T-DNA into scaffold 261 of the flax genome. A) The insertion site of the FP967 TDNA into Norlin gDNA at scaffold261. The event specific assay detects uninterrupted gDNA from Norlin using primers P13 and P14 and Taqman prb3. See Tables 1 and 2 for more details. B) Known (in colour) and unknown (opaque grey) portions of the FP967 TDNA at the beginning of the project. The sequences and orientations of the LB and flanking region, the pBR322 fragments and the LIH had not been confirmed. The construct specific assay, which detects the DHFR fragment form E. coli and the Nos terminator, is indicated (P3, P4 and prb2). The event specific assay, developed in this project, is also shown. It uses a primer in Norlin gDNA (P13 or P14), a primer in the RB (P15) and a probe in the RB (prb5) to detect the TDNA. C) Deduced T-DNA structure after NGS and PCR fragment cloning. The inverted portion of the TDNA inserted between FFS1 and the LB is indicated, as is the new event-specific assay, which spans the junction between the SAT2 gene of the SpecR cassette and the LB (P85, P86 and prb28). The orientation and sequence of the LIH, AtALS, NPTII, SpecR cassette and internal Nos gene were deduced. Inverted sections were found to be oriented in the reverse direction.
    Figure Legend Snippet: Cartoon of the insertion of the FP967 T-DNA into scaffold 261 of the flax genome. A) The insertion site of the FP967 TDNA into Norlin gDNA at scaffold261. The event specific assay detects uninterrupted gDNA from Norlin using primers P13 and P14 and Taqman prb3. See Tables 1 and 2 for more details. B) Known (in colour) and unknown (opaque grey) portions of the FP967 TDNA at the beginning of the project. The sequences and orientations of the LB and flanking region, the pBR322 fragments and the LIH had not been confirmed. The construct specific assay, which detects the DHFR fragment form E. coli and the Nos terminator, is indicated (P3, P4 and prb2). The event specific assay, developed in this project, is also shown. It uses a primer in Norlin gDNA (P13 or P14), a primer in the RB (P15) and a probe in the RB (prb5) to detect the TDNA. C) Deduced T-DNA structure after NGS and PCR fragment cloning. The inverted portion of the TDNA inserted between FFS1 and the LB is indicated, as is the new event-specific assay, which spans the junction between the SAT2 gene of the SpecR cassette and the LB (P85, P86 and prb28). The orientation and sequence of the LIH, AtALS, NPTII, SpecR cassette and internal Nos gene were deduced. Inverted sections were found to be oriented in the reverse direction.

    Techniques Used: Construct, Next-Generation Sequencing, Polymerase Chain Reaction, Clone Assay, Sequencing

    56) Product Images from "A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples"

    Article Title: A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples

    Journal: BMC Genetics

    doi: 10.1186/1471-2156-7-32

    PCR amplification of DNA polymorphisms from aged sunfish tissues, employing Lma20 primers to amplify microsatellite regions. Lane 1: molecular weight markers (Hi-Lo Marker, Minnesota Molecular), size indicated in basepairs. Lanes 2, 4, 6, 8, and 10: PCR amplification of sunfish DNA from tissues that were stored in 100% ethanol. Lanes 3, 5, 7, 9, and 11: PCR amplification of sunfish DNA from tissues that were stored in a dried state. The period of aging is indicated for each set of samples. 2% agarose gel stained with ethidium bromide.
    Figure Legend Snippet: PCR amplification of DNA polymorphisms from aged sunfish tissues, employing Lma20 primers to amplify microsatellite regions. Lane 1: molecular weight markers (Hi-Lo Marker, Minnesota Molecular), size indicated in basepairs. Lanes 2, 4, 6, 8, and 10: PCR amplification of sunfish DNA from tissues that were stored in 100% ethanol. Lanes 3, 5, 7, 9, and 11: PCR amplification of sunfish DNA from tissues that were stored in a dried state. The period of aging is indicated for each set of samples. 2% agarose gel stained with ethidium bromide.

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker, Agarose Gel Electrophoresis, Staining

    PCR products of bluegill DNA (Lake Wapalanne), employing Lma20 primers to amplify microsatellite regions. Lane 1: molecular weight markers (Hi-Lo Marker, Minnesota Molecular), size indicated in basepairs. Lanes 2–7: bluegill Lma20 microsatellite polymorphisms. Buccal tissues employed for this experiment were stored overnight in 100% ethanol before DNA extraction after 24 hours. Heterozygotes and homozygotes for the Lma20 marker are clearly delineated in the individual fish. 2% agarose gel stained with ethidium bromide. The image was inverted to a negative by Scion computer software (Scion, Inc., Frederick, Maryland).
    Figure Legend Snippet: PCR products of bluegill DNA (Lake Wapalanne), employing Lma20 primers to amplify microsatellite regions. Lane 1: molecular weight markers (Hi-Lo Marker, Minnesota Molecular), size indicated in basepairs. Lanes 2–7: bluegill Lma20 microsatellite polymorphisms. Buccal tissues employed for this experiment were stored overnight in 100% ethanol before DNA extraction after 24 hours. Heterozygotes and homozygotes for the Lma20 marker are clearly delineated in the individual fish. 2% agarose gel stained with ethidium bromide. The image was inverted to a negative by Scion computer software (Scion, Inc., Frederick, Maryland).

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, DNA Extraction, Fluorescence In Situ Hybridization, Agarose Gel Electrophoresis, Staining, Software

    57) Product Images from "Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process"

    Article Title: Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt1154

    CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).
    Figure Legend Snippet: CRISPR adaptation to HHPV-2 infection. ( A ) Depiction of the single CRISPR structure and the preceding cas operon carried by the H. hispanica ATCC 33960 genome. Primers used to examine CRISPR expansion (in panel B) are shown as black arrows and listed in Supplementary Table S2 . ( B ) PCR assay to detect CRISPR expansion at the leader end (L1–L2), the inner part (I1–I2) or the distal end (D1–D2). DNA sampled from infected (+) or uninfected (−) cells was used as PCR templates. Lane M, dsDNA size marker. ( C ) The sequence logo showing the conserved PAM of TTC. The 20 nt upstream of each protospacer observed during HHPV-2 infection were collected and analyzed with WebLogo ( http://weblogo.berkeley.edu/logo.cgi ).

    Techniques Used: CRISPR, Infection, Polymerase Chain Reaction, Marker, Sequencing

    Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.
    Figure Legend Snippet: Adaptation to HHPV-2 infection under different cas genetic backgrounds. ( A ) Cas requirement for adaptation. For each cas mutant, DNA was sampled from cells transformed with an empty plasmid (−) or the plasmid carrying the deleted cas gene(s) (+). The plasmid-carried cas gene(s) was/were under the control of the cas operon promoter. ( B ) Requirements for the nuclease and helicase activities of Cas3. Alanine replacement was performed for the putative key residues in the HD nuclease domain (H20A, H55A, D56A and D229A) and the DExD/H helicase domain (K315A, D439A and E440A). Another two conserved residues (His6 and Lys113) were also mutated. The empty plasmid (−) and the plasmid carrying a wild-type Cas3 (Cas3 WT ) were used, respectively, as negative and positive controls. Lane Ms, dsDNA size markers.

    Techniques Used: Infection, Mutagenesis, Transformation Assay, Plasmid Preparation, Mass Spectrometry

    58) Product Images from "A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA"

    Article Title: A new way of measuring apoptosis by absolute quantitation of inter-nucleosomally fragmented genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks334

    ApoqPCR utility in an in vitro context. ( a ) ApoqPCR can consistently measure apoptosis down to sub-100 cell-equivalent levels. Four-fold dilutions of PBMC gDNA from each of four individuals were constructed and pg apoptotic DNA/1000 cells determined in three experiments for all dilutions, yielding nine final values per dilution. Cell-equivalent values were determined by Cell Number qPCR. Error bars ± 1 SEM; P -values determined by Student’s t -test comparing final values for the highest dilution against the lowest dilution. Final values are graphed when values determined by qLM–PCR or Cell Number qPCR arose within the limits of each of the two standard curves, with the exception of final (bracketed) values obtained for clinical sample 4, where, because of this sample’s very low fragmentation level, qLM–PCR values arose below the standard curve despite reasonable cell numbers. This exception is shown to illustrate consistency at this very low apoptosis level (graph inset) and to indicate that it may be possible to extend the lower limits of the qLM–PCR standard curve. ( b ) and ( c ) Using Jurkat cells, ApoqPCR values are concordant with apoptosis- (TUNEL-) positive cells counted by fluorescence microscopy when working with minute samples (≤100 cell equivalents). Three independent experiments were combined. Table gives values obtained using either [mean cells counted per microscope field] or [mean cells worth of gDNA], each mean being
    Figure Legend Snippet: ApoqPCR utility in an in vitro context. ( a ) ApoqPCR can consistently measure apoptosis down to sub-100 cell-equivalent levels. Four-fold dilutions of PBMC gDNA from each of four individuals were constructed and pg apoptotic DNA/1000 cells determined in three experiments for all dilutions, yielding nine final values per dilution. Cell-equivalent values were determined by Cell Number qPCR. Error bars ± 1 SEM; P -values determined by Student’s t -test comparing final values for the highest dilution against the lowest dilution. Final values are graphed when values determined by qLM–PCR or Cell Number qPCR arose within the limits of each of the two standard curves, with the exception of final (bracketed) values obtained for clinical sample 4, where, because of this sample’s very low fragmentation level, qLM–PCR values arose below the standard curve despite reasonable cell numbers. This exception is shown to illustrate consistency at this very low apoptosis level (graph inset) and to indicate that it may be possible to extend the lower limits of the qLM–PCR standard curve. ( b ) and ( c ) Using Jurkat cells, ApoqPCR values are concordant with apoptosis- (TUNEL-) positive cells counted by fluorescence microscopy when working with minute samples (≤100 cell equivalents). Three independent experiments were combined. Table gives values obtained using either [mean cells counted per microscope field] or [mean cells worth of gDNA], each mean being

    Techniques Used: In Vitro, Construct, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, TUNEL Assay, Fluorescence, Microscopy

    Molecular process of ligation-mediated PCR. Non-phosphorylated oligonucleotides are annealed then blunt-end ligated to target apoptotic DNA within the gDNA population. Heating to 94°C releases the unligated 12-mers and dissociates the monoclonal antibody from Taq polymerase, allowing synthesis at 72°C of the complement of the 24-mer sequence. Subsequent cycles at 94°C then 72°C allows amplification of target DNA using only the 24-mer as PCR primer. Blue boxes: single strands of target DNA; yellow boxes: 12-mers; orange and red boxes: 24-mers and their synthesized complement respectively. Process modified from Staley et al . ( 17 ).
    Figure Legend Snippet: Molecular process of ligation-mediated PCR. Non-phosphorylated oligonucleotides are annealed then blunt-end ligated to target apoptotic DNA within the gDNA population. Heating to 94°C releases the unligated 12-mers and dissociates the monoclonal antibody from Taq polymerase, allowing synthesis at 72°C of the complement of the 24-mer sequence. Subsequent cycles at 94°C then 72°C allows amplification of target DNA using only the 24-mer as PCR primer. Blue boxes: single strands of target DNA; yellow boxes: 12-mers; orange and red boxes: 24-mers and their synthesized complement respectively. Process modified from Staley et al . ( 17 ).

    Techniques Used: Ligation, Polymerase Chain Reaction, Sequencing, Amplification, Synthesized, Modification

    ( a–c ) Comparison and validation of ApoqPCR against other quantifiers of apoptosis. Log-phase Jurkat cells were incubated with 0, 2 or 20 µM of the topoisomerase I inhibitor camptothecin with cells removed and processed at 0, 1, 2, 3, 4 and 5 h. Apoptosis was measured on the one cell fraction at each time-point. Filled triangle, filled circle, filled square and filled inverted triangle define shifts in values due to 0, 2 and 20 µM camptothecin and vehicle respectively. Error bars ±1 SEM. ( a ) Changes in Jurkat cell apoptotic DNA levels over time as measured by ApoqPCR; 3 experiments where each experiment is a set of annealing/ligation reactions, qLM–PCR reactions and Cell Number qPCR reactions, generating nine replicates (see text). ( b ) Changes in Jurkat cell TUNEL-positivity with time as measured by flow cytometry; four measurements at each plotted value. At each measurement at least 10 000 events were sorted. ( c ) Changes in Jurkat cell active caspase-3 levels by ELISA; three independent experiments generating six replicates at each plotted value. ( d–f ) ApoqPCR exhibits competitive advantages in sensitivity when assessing low biologically relevant levels of apoptosis. In a separate series of independent experiments, changes in PBMC apoptotic DNA levels induced by small incremental increases in camptothecin dose were quantified in parallel by ( d ) ApoqPCR, ( e ) TUNEL/FACS and ( f ) Annexin V+7AAD/FACS. For each flow cytometry measurement at least 10 000 events were sorted. Bars ±1 SEM, n = 6 over two experiments; see also Supplementary Figure S5 .
    Figure Legend Snippet: ( a–c ) Comparison and validation of ApoqPCR against other quantifiers of apoptosis. Log-phase Jurkat cells were incubated with 0, 2 or 20 µM of the topoisomerase I inhibitor camptothecin with cells removed and processed at 0, 1, 2, 3, 4 and 5 h. Apoptosis was measured on the one cell fraction at each time-point. Filled triangle, filled circle, filled square and filled inverted triangle define shifts in values due to 0, 2 and 20 µM camptothecin and vehicle respectively. Error bars ±1 SEM. ( a ) Changes in Jurkat cell apoptotic DNA levels over time as measured by ApoqPCR; 3 experiments where each experiment is a set of annealing/ligation reactions, qLM–PCR reactions and Cell Number qPCR reactions, generating nine replicates (see text). ( b ) Changes in Jurkat cell TUNEL-positivity with time as measured by flow cytometry; four measurements at each plotted value. At each measurement at least 10 000 events were sorted. ( c ) Changes in Jurkat cell active caspase-3 levels by ELISA; three independent experiments generating six replicates at each plotted value. ( d–f ) ApoqPCR exhibits competitive advantages in sensitivity when assessing low biologically relevant levels of apoptosis. In a separate series of independent experiments, changes in PBMC apoptotic DNA levels induced by small incremental increases in camptothecin dose were quantified in parallel by ( d ) ApoqPCR, ( e ) TUNEL/FACS and ( f ) Annexin V+7AAD/FACS. For each flow cytometry measurement at least 10 000 events were sorted. Bars ±1 SEM, n = 6 over two experiments; see also Supplementary Figure S5 .

    Techniques Used: Incubation, Ligation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Cytometry, Enzyme-linked Immunosorbent Assay, FACS

    Merging ligation-mediated PCR with qPCR (qLM–PCR) to generate an apoptotic DNA standard curve. Apoptotic gDNA defined as complete was measured spectrophotometrically, diluted to cover a 1000-fold range of quantities and added to annealing/ligation reactions. Aliquots of diluted annealing/ligation reactions were added to qLM–PCR reactions and fluorescence of triplicates monitored in real time. ( a ) Mean of triplicate amplification plots shown. NTC: annealing/ligation with no gDNA into a qLM–PCR reaction as a no template control. ( b ) Representative standard curve showing correlation between C t and amount of apoptotic DNA. Analysis of inter-run apoptotic standard curve consistency is reported in Supplementary Table S2a and S2b . ( c ) Electrophoresis on one agarose gel of actual qLM–PCR reactions (halted at cycle 20) using 2-fold dilutions of completely apoptotic DNA after ethidium bromide staining and de-staining. Fifty percent of each 25 µl reaction per well. M: 500 ng molecular weight markers per well with sizes shown on the right. Tracks 1 and 14: not loaded. Track 13: NTC. Track 2 to 12: 2-fold dilutions of complete apoptotic DNA from 15 000 pg to 14.6 pg in each qLM–PCR reaction. Gel shows that the qLM–PCR standard curve is generated by amplification of apoptotic fragments.
    Figure Legend Snippet: Merging ligation-mediated PCR with qPCR (qLM–PCR) to generate an apoptotic DNA standard curve. Apoptotic gDNA defined as complete was measured spectrophotometrically, diluted to cover a 1000-fold range of quantities and added to annealing/ligation reactions. Aliquots of diluted annealing/ligation reactions were added to qLM–PCR reactions and fluorescence of triplicates monitored in real time. ( a ) Mean of triplicate amplification plots shown. NTC: annealing/ligation with no gDNA into a qLM–PCR reaction as a no template control. ( b ) Representative standard curve showing correlation between C t and amount of apoptotic DNA. Analysis of inter-run apoptotic standard curve consistency is reported in Supplementary Table S2a and S2b . ( c ) Electrophoresis on one agarose gel of actual qLM–PCR reactions (halted at cycle 20) using 2-fold dilutions of completely apoptotic DNA after ethidium bromide staining and de-staining. Fifty percent of each 25 µl reaction per well. M: 500 ng molecular weight markers per well with sizes shown on the right. Tracks 1 and 14: not loaded. Track 13: NTC. Track 2 to 12: 2-fold dilutions of complete apoptotic DNA from 15 000 pg to 14.6 pg in each qLM–PCR reaction. Gel shows that the qLM–PCR standard curve is generated by amplification of apoptotic fragments.

    Techniques Used: Ligation, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Fluorescence, Amplification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Molecular Weight, Generated

    Production and verification of completely apoptotic DNA. ( a ) After 5 h incubation, Jurkat cell gDNA was purified as in ‘Materials and Methods’ section, electrophoresed together on a 1.5% agarose gel, stained with ethidium bromide then destained. Tracks 1 and 4: 500 ng per track molecular weight markers (1 kb Plus DNA ladder, Invitrogen). Track 2: 500 ng Jurkat gDNA incubated with 8 µM staurosporine for 5 h. Track 3:500 ng of untreated (staurosporine-negative) Jurkat gDNA. ( b ) Trace intensities of samples in tracks 2 and 3 from gel of (a). The location in the figure of the y-axis and the length of the x-axis are aligned with the gel in (a). The track 2 trace reveals a complete absence of high molecular weight (unfragmented) gDNA, hence defined as completely (‘100%’) apoptotic DNA. ( c ) The extent of apoptosis was verified by flow cytometric measurement of TUNEL-positivity of cells from the same experiment shown in (a) and (b). Gating excluded sub-cellular debris and selected the total cell population. At least 10 000 events were sorted at each measurement. Black line histogram: negative control cells after 5 h incubation (TUNEL label, no enzyme); autofluorescence controls showed peaks at equivalent positions to negative control (data not shown); grey tint histogram: three replicates of untreated cells after 5 h; red tint histogram: three replicates of cells treated with 8 µM staurosporine for 5 h. Mean TUNEL-positivity was 99.3%. To confirm reproducibility of generating completely apoptotic DNA, this experiment was repeated twice on separate occasions with comparable results ( Supplementary Figure S3 ).
    Figure Legend Snippet: Production and verification of completely apoptotic DNA. ( a ) After 5 h incubation, Jurkat cell gDNA was purified as in ‘Materials and Methods’ section, electrophoresed together on a 1.5% agarose gel, stained with ethidium bromide then destained. Tracks 1 and 4: 500 ng per track molecular weight markers (1 kb Plus DNA ladder, Invitrogen). Track 2: 500 ng Jurkat gDNA incubated with 8 µM staurosporine for 5 h. Track 3:500 ng of untreated (staurosporine-negative) Jurkat gDNA. ( b ) Trace intensities of samples in tracks 2 and 3 from gel of (a). The location in the figure of the y-axis and the length of the x-axis are aligned with the gel in (a). The track 2 trace reveals a complete absence of high molecular weight (unfragmented) gDNA, hence defined as completely (‘100%’) apoptotic DNA. ( c ) The extent of apoptosis was verified by flow cytometric measurement of TUNEL-positivity of cells from the same experiment shown in (a) and (b). Gating excluded sub-cellular debris and selected the total cell population. At least 10 000 events were sorted at each measurement. Black line histogram: negative control cells after 5 h incubation (TUNEL label, no enzyme); autofluorescence controls showed peaks at equivalent positions to negative control (data not shown); grey tint histogram: three replicates of untreated cells after 5 h; red tint histogram: three replicates of cells treated with 8 µM staurosporine for 5 h. Mean TUNEL-positivity was 99.3%. To confirm reproducibility of generating completely apoptotic DNA, this experiment was repeated twice on separate occasions with comparable results ( Supplementary Figure S3 ).

    Techniques Used: Incubation, Purification, Agarose Gel Electrophoresis, Staining, Molecular Weight, Flow Cytometry, TUNEL Assay, Negative Control

    59) Product Images from "Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells"

    Article Title: Ehrlichia chaffeensis Uses Its Surface Protein EtpE to Bind GPI-Anchored Protein DNase X and Trigger Entry into Mammalian Cells

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003666

    EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P
    Figure Legend Snippet: EtpE-C is exposed at the bacterial surface, and anti-EtpE-C neutralizes E. chaffeensis infection in vitro . (A) Western blot analysis of E. chaffeensis- infected ( Ech ) and uninfected DH82 cells at 60 h pi using anti-EtpE-N (α-EtpE-N) and anti-EtpE-C (α-EtpE-C). (B) Double immunofluorescence labeling of E. chaffeensis- infected human primary macrophages derived from peripheral blood monocytes at 56 h pi. Cells were fixed with PFA, permeabilized with saponin, and labeled with anti-EtpE-C and anti- E. chaffeensis major outer membrane protein P28. The white dashed line denotes the macrophage contour. The boxed region indicates the area enlarged in the smaller panels to the right. Merge/DIC: Fluorescence images merged with Differential interference contrast image (DIC). A single z -plane (0.4 µm thickness) by deconvolution microscopy was shown. Scale bar, 2 µm. (C) E. chaffeensis was incubated with DH82 cells for 30 min and double immunofluorescence labeling was performed using anti-EtpE-C and anti- E. chaffeensis P28 without permeabilization. DAPI was used to label DNA. Scale bar, 1 µm (see also suppl. Fig. S2 ). (D) Numbers of E. chaffeensis bound to RF/6A cells at 30 min pi. Host cell-free E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and incubated with RF/6A cells for 30 min. Unbound E. chaffeensis was washed away, cells were fixed with PFA, and E. chaffeensis labeled with anti-P28 without permeabilization. E. chaffeensis in 100 cells were scored. (E) Numbers of E. chaffeensis internalized into RF/6A cells at 2 h pi. E. chaffeensis was pretreated with anti-rEtpE-C or preimmune mouse serum and incubated with RF/6A cells for 2 h. To distinguish intracellular from bound E. chaffeensis , unbound E. chaffeensis was washed away and cells were processed for two rounds of immunostaining with anti-P28; first without permeabilization to detect bound but not internalized E. chaffeensis (AF555–conjugated secondary antibody) and second round with saponin permeabilization to detect total E. chaffeensis , i.e., bound plus internalized (AF488–conjugated secondary antibody). E. chaffeensis in 100 cells was scored. The black bar represents total E. chaffeensis and the white bar represents internalized E. chaffeensis (total minus bound) (see also suppl. Fig. S3 ). (F) Infection of RF/6A cells with E. chaffeensis at 48 h pi. E. chaffeensis was pretreated with anti-EtpE-C or preimmune mouse serum and used to infect RF/6A cells; cells were harvested at 48 h pi. qPCR for E. chaffeensis 16S rDNA was normalized with G3PDH DNA. Data represent the mean and standard deviation of triplicate samples and are representative of three independent experiments. *Significantly different ( P

    Techniques Used: Infection, In Vitro, Western Blot, Immunofluorescence, Labeling, Derivative Assay, Fluorescence, Microscopy, Incubation, Immunostaining, Real-time Polymerase Chain Reaction, Standard Deviation

    60) Product Images from "Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker"

    Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-24

    MSP results of two positive controls . Lane 1; MSP product of universally methylated DNA by applying methylated cytosine specific primers. Lane 2; the same as lane 1 but unmethylated cytosine specific primers were used. lane 3; MSP product of blood DNA extracted from a healthy donor treated with DNA methylase (CpG methyl transferase) and application of methylated cytosine specific primers, lane 4; the same as lane 3 but unmethylated cytosine specific primers were used.
    Figure Legend Snippet: MSP results of two positive controls . Lane 1; MSP product of universally methylated DNA by applying methylated cytosine specific primers. Lane 2; the same as lane 1 but unmethylated cytosine specific primers were used. lane 3; MSP product of blood DNA extracted from a healthy donor treated with DNA methylase (CpG methyl transferase) and application of methylated cytosine specific primers, lane 4; the same as lane 3 but unmethylated cytosine specific primers were used.

    Techniques Used: Methylation

    61) Product Images from "Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis"

    Article Title: Involvement of Multiple Gene-Silencing Pathways in a Paramutation-like Phenomenon in Arabidopsis

    Journal: Cell reports

    doi: 10.1016/j.celrep.2015.04.034

    Characterization of the Paramutation-like Phenotype and Molecular Features of the pRD29A-LUC Transgene (A–E) Schematic diagrams showing the genetic crosses performed on plants with indicated genotype (black or red italic letter). The luminescence image above each genotype represents the overall LUC phenotype of 45–50 seedlings. Blue letters indicate the generation number of plants used for the analyses: “F” denotes filial generation of crosses; “BC” denotes crosses made with WT+LUC plants; “S” denotes self-crosses. (F) Northern blotting analyses for pRD29A-LUC and endoRD29A in plants listed in (A)–(E). Two-week-old seedlings before (indicated by −) and after (indicated by +) stress treatment were used for the analyses. TUB8 and COR15A serve as the loading control and the stress-response control, respectively. (G) DNA methylation levels of the transgenic RD29A promoter region as examined by bisulfite sequencing.
    Figure Legend Snippet: Characterization of the Paramutation-like Phenotype and Molecular Features of the pRD29A-LUC Transgene (A–E) Schematic diagrams showing the genetic crosses performed on plants with indicated genotype (black or red italic letter). The luminescence image above each genotype represents the overall LUC phenotype of 45–50 seedlings. Blue letters indicate the generation number of plants used for the analyses: “F” denotes filial generation of crosses; “BC” denotes crosses made with WT+LUC plants; “S” denotes self-crosses. (F) Northern blotting analyses for pRD29A-LUC and endoRD29A in plants listed in (A)–(E). Two-week-old seedlings before (indicated by −) and after (indicated by +) stress treatment were used for the analyses. TUB8 and COR15A serve as the loading control and the stress-response control, respectively. (G) DNA methylation levels of the transgenic RD29A promoter region as examined by bisulfite sequencing.

    Techniques Used: Northern Blot, DNA Methylation Assay, Transgenic Assay, Methylation Sequencing

    Multiple Epigenetic Pathways Are Required to Maintain LUC’ Silencing (A) Bioluminescence and bright field imaging results using rosette leaves from mutant+LUC plants. Genotypes of the plants are marked in yellow on the bright field image. The F3 plants used for the analyses were pre-screened for the presence of pRD29A-LUC transgene. (B) Transcript levels of pRD29A-LUC and endoRD29A genes in the F3 mutant plants are examined by northern blotting. Please note that the signals from WT+ LUC plants differ on different blots due to different exposure time, which serve as a positive control. Stress-treated and control plants are indicated by − and +, respectively. TUB4 and COR15A each serve as the loading control and the control for normal cold response. (C) DNA methylation levels at the transgenic RD29A promoter in the mutant + LUC’ crosses F4 plants as measured by bisulfite sequencing. (D) Northern blotting analyses of 24-nt siRNAs generated from the RD29A promoter (endogenous + transgenic). U6 snoRNA and miR167 each serves as the loading control and microRNA pathway control. (E) Chromatin immunoprecipitation followed by quantitative PCR was used to examine histone H3 acetylation and H3K9me2 levels at the transgenic RD29A promoter. Error bars indicate SD calculated from qPCR reactions of three technical replicates.
    Figure Legend Snippet: Multiple Epigenetic Pathways Are Required to Maintain LUC’ Silencing (A) Bioluminescence and bright field imaging results using rosette leaves from mutant+LUC plants. Genotypes of the plants are marked in yellow on the bright field image. The F3 plants used for the analyses were pre-screened for the presence of pRD29A-LUC transgene. (B) Transcript levels of pRD29A-LUC and endoRD29A genes in the F3 mutant plants are examined by northern blotting. Please note that the signals from WT+ LUC plants differ on different blots due to different exposure time, which serve as a positive control. Stress-treated and control plants are indicated by − and +, respectively. TUB4 and COR15A each serve as the loading control and the control for normal cold response. (C) DNA methylation levels at the transgenic RD29A promoter in the mutant + LUC’ crosses F4 plants as measured by bisulfite sequencing. (D) Northern blotting analyses of 24-nt siRNAs generated from the RD29A promoter (endogenous + transgenic). U6 snoRNA and miR167 each serves as the loading control and microRNA pathway control. (E) Chromatin immunoprecipitation followed by quantitative PCR was used to examine histone H3 acetylation and H3K9me2 levels at the transgenic RD29A promoter. Error bars indicate SD calculated from qPCR reactions of three technical replicates.

    Techniques Used: Imaging, Mutagenesis, Northern Blot, Positive Control, DNA Methylation Assay, Transgenic Assay, Methylation Sequencing, Generated, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    The pRD29A-LUC Transgene Is Likely a 13-Copy Repeat (A) Number of RD29A promoter sequences in WT+LUC measured by qPCR. Non-transgenic WT plants were used as a reference of one. Error bars indicate SD calculated from qPCR reactions of three technical replicates. (B) Southern blotting of HindIII- and XbaI-digested genomic DNA using a LUC -specific probe ( Figure S1A ). DNA size markers were indicated on the left side of the membrane. LUC -specific bands were indicated by red triangles.
    Figure Legend Snippet: The pRD29A-LUC Transgene Is Likely a 13-Copy Repeat (A) Number of RD29A promoter sequences in WT+LUC measured by qPCR. Non-transgenic WT plants were used as a reference of one. Error bars indicate SD calculated from qPCR reactions of three technical replicates. (B) Southern blotting of HindIII- and XbaI-digested genomic DNA using a LUC -specific probe ( Figure S1A ). DNA size markers were indicated on the left side of the membrane. LUC -specific bands were indicated by red triangles.

    Techniques Used: Real-time Polymerase Chain Reaction, Transgenic Assay, Southern Blot

    62) Product Images from "A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies"

    Article Title: A novel antisense long noncoding RNA within the IGF1R gene locus is imprinted in hematopoietic malignancies

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku549

    Characterization of IRAIN as an antisense lncNRA. ( A ) The diagram of the IRAIN / IGF1R locus. p IRAIN : IRAIN lncRNA promoter; p IR : IGF1R coding RNA promoter. Vertical arrows: the location of lncRNA PCR primers. ( B and C ) Mapping of the IRAIN lncRNA in K562 leukemia cells. gDNA: genomic DNA used as the control to test the efficiency of the PCR primers. M: 100 bp marker. ( D ) Northern blot of the IRAIN lncRNA in breast cancer tissues. Total RNA from three breast cancer tumors was separated on a 1.5% (w/v) denaturing agarose gel and was hybridized with the 32 P-dCTP labeled IRAIN cDNA clone probe. GAPDH was used as the control. E: IRAIN lncRNA is an antisense lncRNA. Horizontal arrows: SSRT-PCR primers used to map the orientation of IRAIN lncRNA. The strand-specific cDNAs were synthesized using either the 5′- or the 3′-oligonucleotides at sites C, D and F. A pair of PCR primers located between two cDNA oligonucleotides was then used to determine the transcription orientation of the IRAIN lncRNA. M: 100 bp marker; input: total RNA collected before SSRT-PCR; RT: reverse transcriptase.
    Figure Legend Snippet: Characterization of IRAIN as an antisense lncNRA. ( A ) The diagram of the IRAIN / IGF1R locus. p IRAIN : IRAIN lncRNA promoter; p IR : IGF1R coding RNA promoter. Vertical arrows: the location of lncRNA PCR primers. ( B and C ) Mapping of the IRAIN lncRNA in K562 leukemia cells. gDNA: genomic DNA used as the control to test the efficiency of the PCR primers. M: 100 bp marker. ( D ) Northern blot of the IRAIN lncRNA in breast cancer tissues. Total RNA from three breast cancer tumors was separated on a 1.5% (w/v) denaturing agarose gel and was hybridized with the 32 P-dCTP labeled IRAIN cDNA clone probe. GAPDH was used as the control. E: IRAIN lncRNA is an antisense lncRNA. Horizontal arrows: SSRT-PCR primers used to map the orientation of IRAIN lncRNA. The strand-specific cDNAs were synthesized using either the 5′- or the 3′-oligonucleotides at sites C, D and F. A pair of PCR primers located between two cDNA oligonucleotides was then used to determine the transcription orientation of the IRAIN lncRNA. M: 100 bp marker; input: total RNA collected before SSRT-PCR; RT: reverse transcriptase.

    Techniques Used: Polymerase Chain Reaction, Marker, Northern Blot, Agarose Gel Electrophoresis, Labeling, Synthesized

    The IRAIN lncRNA is imprinted in hematopoietic cells. ( A ) Polymorphic restriction enzymes used to distinguish the two parental alleles. SNP: single nucleotide polymorphism. The IRAIN lncRNA was reverse transcribed into cDNA using SSRT oligonucleotides. The PCR products were digested by polymorphic Alu I and Sac II. ( B and C ) Allelic expression of IRAIN lncRNA at the E site in KG-1 and KG-1a leukemia cells. gDNA: heterozygous genomic DNA. Note the single ‘A’ allele or the single ‘G’ allele expression of IRAIN lncRNA. (D) Parental imprinting of IRAIN lncRNA by tracking allelic expression in three families. Genomic DNA and cDNA were amplified from peripheral blood cells and the PCR products were sequenced for the A/G alleles. Note the monoallelic expression of IRAIN lncRNA from the paternal allele.
    Figure Legend Snippet: The IRAIN lncRNA is imprinted in hematopoietic cells. ( A ) Polymorphic restriction enzymes used to distinguish the two parental alleles. SNP: single nucleotide polymorphism. The IRAIN lncRNA was reverse transcribed into cDNA using SSRT oligonucleotides. The PCR products were digested by polymorphic Alu I and Sac II. ( B and C ) Allelic expression of IRAIN lncRNA at the E site in KG-1 and KG-1a leukemia cells. gDNA: heterozygous genomic DNA. Note the single ‘A’ allele or the single ‘G’ allele expression of IRAIN lncRNA. (D) Parental imprinting of IRAIN lncRNA by tracking allelic expression in three families. Genomic DNA and cDNA were amplified from peripheral blood cells and the PCR products were sequenced for the A/G alleles. Note the monoallelic expression of IRAIN lncRNA from the paternal allele.

    Techniques Used: Polymerase Chain Reaction, Expressing, Amplification

    63) Product Images from "Circularization pathway of a bacterial group II intron"

    Article Title: Circularization pathway of a bacterial group II intron

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1381

    Identification of Ll.LtrB 3′ ends in vivo . Free intron 3′ ends were amplified by RT-PCR from L. lactis total RNA extracts. ( A ) Intron 3′ ends were identified by first extending the intron RNA with a polyA tail followed by the synthesis of a cDNA with an oligo dT. The RNA strand was removed by an RNAse H treatment and the single strand DNA amplified by PCR. ( B ) The PCR reactions were ran on a 2% agarose gel and the chromatogram of some of the sequenced bands are shown ( C – D ). The same procedure was repeated for the Ll.LtrB-ΔA construct but extending a polyU instead of a polyA tail at the 3′ end of the intron ( E ).
    Figure Legend Snippet: Identification of Ll.LtrB 3′ ends in vivo . Free intron 3′ ends were amplified by RT-PCR from L. lactis total RNA extracts. ( A ) Intron 3′ ends were identified by first extending the intron RNA with a polyA tail followed by the synthesis of a cDNA with an oligo dT. The RNA strand was removed by an RNAse H treatment and the single strand DNA amplified by PCR. ( B ) The PCR reactions were ran on a 2% agarose gel and the chromatogram of some of the sequenced bands are shown ( C – D ). The same procedure was repeated for the Ll.LtrB-ΔA construct but extending a polyU instead of a polyA tail at the 3′ end of the intron ( E ).

    Techniques Used: In Vivo, Amplification, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Construct

    64) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.
    Figure Legend Snippet: Cyclin A treatment synchronizes DNA replication in Xenopus egg extracts (A–B) To synchronize DNA replication in Xenopus egg extracts, we treated extracts with Cyclin A, which probably accelerates replication initiation 45 . Plasmid DNA was incubated in High Speed Supernatant for 20 minutes, then either buffer or Cyclin A was added for a further 20 minutes. NucleoPlasmic extract was added to initiate DNA replication, along with [α- 32 P]dATP to label replication intermediates. Replication products were separated on a native agarose gel, detected by autoradiography (A), and quantified (B). In the presence of vehicle, replication was not complete by 9.5 minutes, but in the presence of Cyclin A, replication was almost complete by 4.5 minutes (B). Thus, Cyclin A treatment approximately doubles the speed of DNA replication in Xenopus egg extracts. (C–F) To test whether Cyclin A affects the ability of LacR to inhibit termination, we monitored dissolution of plasmids containing a 12x or 16x LacR array in the presence and absence of Cyclin A. p[ lacO x12], p[ lacO x16], and the parental control plasmid p[ empty ] were incubated with LacR, and then treated with buffer or Cyclin A before replication was initiated with NPE in the presence of [α- 32 P]dATP. Samples were withdrawn when dissolution of p[ empty ] plateaued (9.5 minutes in the presence of buffer, 4.5 minutes in the presence of Cyclin A). Given that Cyclin A treatment approximately doubles the speed of replication (see B), samples were withdrawn from these reactions twice as frequently as the buffer-treated samples. To measure dissolution, radiolabelled termination intermediates were cut with XmnI to monitor the conversion of double-Y molecules to linear molecules. Cut molecules were separated on a native agarose gel and detected by autoradiography (C,E). By the time the first sample was withdrawn, dissolution of p[ empty ] was essentially complete, in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Importantly, dissolution of p[ lacO x12] and p[ lacO x16] was prevented in the absence (9.5 min, D) or presence (4.5 min, F) of Cyclin A. Moreover, dissolution occurred approximately twice as fast in the presence of Cyclin A (note the similarity between (D) and (F) even though samples are withdrawn twice as frequently in F.) consistent with replication being approximately twice as fast in the presence of Cyclin A. Therefore, Cyclin A does not affect the ability of a LacR array to block replication forks.

    Techniques Used: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Blocking Assay

    Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.
    Figure Legend Snippet: Replisome progression through 12x and 32x lacO arrays (A–D) To test whether replisomes meet later in a lacO x32 array than a lacO x12 array, we monitored dissolution. LacR Block-IPTG release was performed on p[ lacO x12] and p[ lacO x32] and radiolabelled termination intermediates were digested with XmnI to monitor the conversion of double-Y molecules to linear molecules (Dissolution). Cleaved molecules were separated on a native agarose gel, detected by autoradiography (A,C), and quantified (B,D). Upon IPTG addition, dissolution was delayed by at least 1 minute within the 32x lacO array compared to the 12x lacO array (B,D). Moreover, by 6 minutes, 92% of forks had undergone dissolution on p[ lacO x12] while only 9% had dissolved on p[ lacO x32] (B,D). (E) Stall products within the 12x lacO array ( Fig. 3B , Lane 2) were quantified, signal was corrected based on size differences of the products, and the percentage of stall products at each stall point was calculated. 78% of leading strands stalled at the first three arrest points (red columns), 19% stalled at the fourth to tenth arrest points (yellow columns) and the remaining 3% stalled at the tenth to fourteenth arrest points (grey columns). The appearance of fourteen arrest points reproducible and surprising, given that the presence of only twelve lacO sequences was confirmed by sequencing in the very preparation of p[ lacO x12] that was used in Fig. 3 . The thirteenth and fourteenth arrest points cannot stem from cryptic lacO sites beyond the twelfth lacO site, as this would position the first leftward leading strand stall product ∼90 nucleotides from the lacO array, instead of the observed ∼30 nucleotides (see F-G ). At present, we do not understand the origin of these stall products. (F–G) Progression of leftward leading strands into the array. The same DNA samples used in Fig. 3 were digested with the nicking enzyme Nb.BsrDI, which released leftward leading strands (F), and separated on a denaturing polyacrylamide gel (G). The lacO sites of p[ lacO x12] are highlighted in blue on the sequencing ladder (G), which was generated using the primer JDO109 (Green arrow, F). Green circles indicate two non-specific products of digestion. These products arise because nicking enzyme activity varies between experiments, even under the same conditions. There was no significant difference in the pattern of leftward leading strand progression between the 12x lacO and 32x lacO arrays, as seen for the rightward leading strands ( Fig. 3B ). Specifically, by 5.67 minutes, the majority of leading strands had extended beyond the seventh lacO repeat within lacO x12 (lane 6) and the equivalent region of lacO x32 (lane 18). Therefore, progression of leftward leading strands is unaffected by the presence of an opposing replisome, suggesting that converging replisomes do not stall when they meet.

    Techniques Used: Blocking Assay, Agarose Gel Electrophoresis, Autoradiography, Sequencing, Generated, Activity Assay

    Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.
    Figure Legend Snippet: Supplemental ChIP data (A) Cartoon depicting the LAC, FLK2 and FAR loci, which were used for ChIP. Their precise locations relative to the leftward edge of the lacO array are indicated. The LAC amplicon is present in four copies distributed across the lacOx16 array and three copies distributed across the lacOx12 array. (B–E) p[ lacO x12] was incubated with buffer or LacR and termination was induced at 5 minutes by IPTG addition. MCM7, RPA, CDC45, and Polε ChIP was performed at different time points after IPTG addition but also in the buffer control and no IPTG control. Recovery of FLK2 was measured as a percentage of input DNA. Upon IPTG addition, ChIP signal declined and by 9 minutes was comparable to the buffer control, demonstrating that unloading of replisomes was induced within 4 minutes of IPTG addition. (F) To test whether movement of the replisome into and out of the lacO array could be detected upon IPTG addition, termination was monitored within a lacO array, and we performed ChIP of the leading strand polymerase Polε, which was inferred to move into and out of the array based on the behavior of leading strands during termination ( Extended Data Fig 2B–E ). It was predicted that Polε ChIP at the LAC locus should increase slightly as Polε enters the lacO array and decline again as converging polymerases pass each other, but persist at FLK2 while the polymerases move out of the array. Prior to IPTG addition, Polε was enriched at LAC and FLK2 compared to FAR , consistent with the leading strands being positioned on either side of the lacO array ( Extended Data Fig 2C , Fig. 3 ). Upon IPTG addition, Polε became modestly enriched at LAC compared to FLK2 (5.5 min) but then declined to similar levels at both LAC and FLK2 by 6.5 min. These data are consistent with the leading strand polymerases entering the lacO array and passing each other. (G–H) To test whether CMG exhibited the same ChIP profile as Polε, MCM7 and CDC45 ChIP was performed using the same samples. Following IPTG addition, MCM7 and CDC45 were enriched at LAC compared to FLK2 (5.5 min), then declined to similar levels at both LAC and FLK2 by 6.5 min, as seen for Polε (F). These data are consistent with a model in which CMGs enter the array and pass each other during termination. A caveat of these experiments is the relatively high recovery of the FAR locus in MCM7, CDC45, and Polε ChIP. Specifically, signal was at most only ∼2-fold enriched at LAC compared to FAR . This was not due to high background binding, because by the end of the experiment (10 minute time point, not shown), we observed a decrease in signal of ∼5–7 fold. Furthermore, we observed ∼5–7 fold enrichment in binding (ChIP) of replisome components to p[ lacO x12] that had been incubated in LacR compared to a buffer control (see G-I, below). Instead, the high FAR signal was likely due to poor spatial resolution of the ChIP. Consistent with this, when a plasmid containing a DNA interstrand cross-link (ICL) was replicated, essentially all replisomes converged upon the ICL but the ChIP signal for MCM7 and CDC45 was only ∼3–4 fold enriched at the ICL compared to a control locus 41 . We speculate that the higher background observed at the control locus in our experiments is due to the decreased distance of the control locus from the experimental locus (1.3 kb for p[ lacO x16] and p[ lacO x12] vs. 2.4 kb for the ICL plasmid) and possibly due to increased catenation of the parental strands during termination. The high signal at FAR should not complicate interpretation of the MCM7, CDC45 and Polε ChIP (F), as signal at FAR was essentially unaltered between 5 and 6.5 minutes. Further evidence that the high signal seen at the FAR locus emanates from forks stalled near the lacO array is presented in panel ( K ). (I) ChIP of RPA was performed on the same chromatin samples used in B-D. As seen for pol ɛ, MCM7, and CDC45, enrichment of RPA at LAC compared to FAR was relatively low, consistent with poor spatial resolution. (J) Predicted binding of CMGs to the LAC, FLK2 and FAR loci before and after IPTG addition if CMGs converging CMG pass each other. (K) To determine whether most forks stalled at the array and not elsewhere in the plasmid, we performed a time course in which p[ lacO x16] undergoing termination was examined by 2-dimensional gel electrophoresis (2-D gel) at various time points. p[ lacO x16] was pre-bound to LacR and replicated in Xenopus egg extract containing [α- 32 P]dATP. Termination was induced by IPTG addition and samples were withdrawn at different times. Radiolabelled replication intermediates were cleaved with XmnI (as in Extended Data Fig. 1A ) and separated according to size and shape on 2-D gels 50 . A parallel reaction was performed in which samples were analyzed by ChIP, which was one of the repeats analyzed in (B)-(E). In the presence of LacR, a subset of Double Y molecules accumulated (blue arrowhead), demonstrating that 83% of replication intermediates (signal in dashed blue circle) contained two forks converged at a specific locus. Following IPTG addition, linear molecules rapidly accumulated (orange arrow) as dissolution occurred. Importantly, the vast majority of signal was present in the discrete double-Y and linear species (blue and orange arrows), demonstrating that the relatively high ChIP signal observed at FAR in panels F-I was derived from forks present at the lacO x16 array and not elsewhere.

    Techniques Used: Chromatin Immunoprecipitation, Amplification, Incubation, Recombinase Polymerase Amplification, Binding Assay, Plasmid Preparation, Nucleic Acid Electrophoresis, Derivative Assay

    The rate of total DNA synthesis does not slow before dissolution (A–C) To further test whether replication stalls or slows prior to dissolution, p[ lacO x12] was pre-incubated with LacR and replicated in Xenopus egg extracts. Termination was then induced by addition of IPTG after 5 minutes. Simultaneously, [α- 32 P]dATP was added to specifically radiolabel DNA synthesized following IPTG addition (A). Radiolabelled DNA was then separated on a native agarose gel and total signal was measured by autoradiography (B). Tota l signal was quantified, normalized to peak signal, and graphed alongside the rate of dissolution, which was also measured in the same experiment (C). This approach gives a highly sensitive measure of DNA synthesis without manipulation of DNA samples. DNA synthesis should occur primarily within the lacO array (see Extended Data Fig. 1 ). Upon IPTG addition, there was an approximately linear increase in signal, which plateaued by 5.83 min. Importantly, dissolution was 65% complete by 5.83 min. Therefore, the large majority of dissolution occurs without stalling of DNA synthesis. (D–E) Experimental repeats of (B–C) (F) The experiments shown in (C–E) were graphed together with mean±s.d. Synthesis data was normalized so that for each experiment, synthesis at 1 min was assigned a value of 84.4%, since this was the average value from (C–D), where synthesis was allowed to plateau. Given the rate of replication fork progression in these egg extracts (260 bp/minute 32 ) and the size of the array (365 bp), forks should require, on average, 0.7 minutes to converge if no stalling occurs (365 bp ÷ 2 ÷ 260 bp/min = 0.7 min). The time required for dissolution was not appreciably longer than this (dissolution was 50% complete by 0.67 min after IPTG addition, (F)), consistent with a lack of stalling. (G–H) The experiment shown in (B–C) was repeated using p[ lacO x16]. Synthesis was approximately linear until 6.17 minutes, at which point 81% of molecules had dissolved, further demonstrating that the majority of dissolution occurs without stalling of DNA synthesis.
    Figure Legend Snippet: The rate of total DNA synthesis does not slow before dissolution (A–C) To further test whether replication stalls or slows prior to dissolution, p[ lacO x12] was pre-incubated with LacR and replicated in Xenopus egg extracts. Termination was then induced by addition of IPTG after 5 minutes. Simultaneously, [α- 32 P]dATP was added to specifically radiolabel DNA synthesized following IPTG addition (A). Radiolabelled DNA was then separated on a native agarose gel and total signal was measured by autoradiography (B). Tota l signal was quantified, normalized to peak signal, and graphed alongside the rate of dissolution, which was also measured in the same experiment (C). This approach gives a highly sensitive measure of DNA synthesis without manipulation of DNA samples. DNA synthesis should occur primarily within the lacO array (see Extended Data Fig. 1 ). Upon IPTG addition, there was an approximately linear increase in signal, which plateaued by 5.83 min. Importantly, dissolution was 65% complete by 5.83 min. Therefore, the large majority of dissolution occurs without stalling of DNA synthesis. (D–E) Experimental repeats of (B–C) (F) The experiments shown in (C–E) were graphed together with mean±s.d. Synthesis data was normalized so that for each experiment, synthesis at 1 min was assigned a value of 84.4%, since this was the average value from (C–D), where synthesis was allowed to plateau. Given the rate of replication fork progression in these egg extracts (260 bp/minute 32 ) and the size of the array (365 bp), forks should require, on average, 0.7 minutes to converge if no stalling occurs (365 bp ÷ 2 ÷ 260 bp/min = 0.7 min). The time required for dissolution was not appreciably longer than this (dissolution was 50% complete by 0.67 min after IPTG addition, (F)), consistent with a lack of stalling. (G–H) The experiment shown in (B–C) was repeated using p[ lacO x16]. Synthesis was approximately linear until 6.17 minutes, at which point 81% of molecules had dissolved, further demonstrating that the majority of dissolution occurs without stalling of DNA synthesis.

    Techniques Used: DNA Synthesis, Incubation, Synthesized, Agarose Gel Electrophoresis, Autoradiography

    65) Product Images from "Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists"

    Article Title: Boosting Wnt activity during colorectal cancer progression through selective hypermethylation of Wnt signaling antagonists

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-14-891

    Image plot showing CpG methylation data from 264 DNA samples as assessed by pyrosequencing. Samples are grouped by pathological category from normal (bottom) to carcinoma (top). Rows represent individual samples. LRN : low-risk normal mucosa from patients with no history of CRC; HRN : high-risk normal mucosa from patients with CRC; HP : hyperplastic polyps; Ad : adenomas; pT : primary colorectal adenocarcinomas and M : metastatic adenocarcinomas to the liver. Columns show the methylation data for each of the CpG dinucleotides analyzed grouped by gene. A scale shown on the right side of the figure represents the colour spectrum reflecting the percentage of CpG methylation as detected by pyrosequencing. White spaces within the plot indicate missing values due to failure of samples to meet bisulfite conversion or pyrosequencing controls or due to lack of DNA. The subset of CIMP and MSI positive primary colorectal carcinomas is highlighted on the plot.
    Figure Legend Snippet: Image plot showing CpG methylation data from 264 DNA samples as assessed by pyrosequencing. Samples are grouped by pathological category from normal (bottom) to carcinoma (top). Rows represent individual samples. LRN : low-risk normal mucosa from patients with no history of CRC; HRN : high-risk normal mucosa from patients with CRC; HP : hyperplastic polyps; Ad : adenomas; pT : primary colorectal adenocarcinomas and M : metastatic adenocarcinomas to the liver. Columns show the methylation data for each of the CpG dinucleotides analyzed grouped by gene. A scale shown on the right side of the figure represents the colour spectrum reflecting the percentage of CpG methylation as detected by pyrosequencing. White spaces within the plot indicate missing values due to failure of samples to meet bisulfite conversion or pyrosequencing controls or due to lack of DNA. The subset of CIMP and MSI positive primary colorectal carcinomas is highlighted on the plot.

    Techniques Used: CpG Methylation Assay, Methylation

    66) Product Images from "TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation"

    Article Title: TET-mediated oxidation of methylcytosine causes TDG or NEIL glycosylase dependent gene reactivation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku552

    In vitro oxidation of mC causes gene reactivation in ESCs. ( a ) Schematic representation of in vitro reporter DNA modification: Unmethylated pOct4-reporter DNA was methylated using the CpG methyltransferase M.SssI. Incubation with purified TET1CD results in oxidation of mC sites to hmC, fC and caC. ( b ) M.SssI treatment of pOct4-mCherry results in full methylation as shown after restriction with the methylation sensitive enzyme HpaII. MspI cuts irrespective of the methylation state. The hmC-specific restriction endonuclease PvuRts1I detects increasing hmC levels during incubation of methylated pOct4-mCherry with TET1CD. ( c ) Cytosine modification states of untreated, methylated and TET1CD oxidized pOct4-mCherry plasmid DNA were detected by slot blot. A 2-fold serial dilution of the plasmid DNA was loaded and detected using antibodies against mC, hmC, fC and caC. A gradual increase of hmC, fC and caC signals was obtained with longer incubation time with TET1CD while the mC signal decreases accordingly. ( d ) Quantification of the slot blot signals of pOct4-mCherry after treatment with TET1CD shows increasing oxidation of mC to hmC, fC and caC. The sum of all CpG modification signals was set to 100%. Error bars indicate standard deviation ( n = 3). ( e ) ESCs were transfected with pOct4-mCherry plasmids containing either unmodified (CpG), methylated (mCpG), TET1CD-oxidized ( ox CpG) or TET1CD mut -treated ( ox *CpG) cytosines. Confocal imaging and quantification show reporter gene silencing upon methylation and reactivation upon oxidation. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. (Right: n = 200 000; error bars indicate standard deviation).
    Figure Legend Snippet: In vitro oxidation of mC causes gene reactivation in ESCs. ( a ) Schematic representation of in vitro reporter DNA modification: Unmethylated pOct4-reporter DNA was methylated using the CpG methyltransferase M.SssI. Incubation with purified TET1CD results in oxidation of mC sites to hmC, fC and caC. ( b ) M.SssI treatment of pOct4-mCherry results in full methylation as shown after restriction with the methylation sensitive enzyme HpaII. MspI cuts irrespective of the methylation state. The hmC-specific restriction endonuclease PvuRts1I detects increasing hmC levels during incubation of methylated pOct4-mCherry with TET1CD. ( c ) Cytosine modification states of untreated, methylated and TET1CD oxidized pOct4-mCherry plasmid DNA were detected by slot blot. A 2-fold serial dilution of the plasmid DNA was loaded and detected using antibodies against mC, hmC, fC and caC. A gradual increase of hmC, fC and caC signals was obtained with longer incubation time with TET1CD while the mC signal decreases accordingly. ( d ) Quantification of the slot blot signals of pOct4-mCherry after treatment with TET1CD shows increasing oxidation of mC to hmC, fC and caC. The sum of all CpG modification signals was set to 100%. Error bars indicate standard deviation ( n = 3). ( e ) ESCs were transfected with pOct4-mCherry plasmids containing either unmodified (CpG), methylated (mCpG), TET1CD-oxidized ( ox CpG) or TET1CD mut -treated ( ox *CpG) cytosines. Confocal imaging and quantification show reporter gene silencing upon methylation and reactivation upon oxidation. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. (Right: n = 200 000; error bars indicate standard deviation).

    Techniques Used: In Vitro, Modification, Methylation, Incubation, Purification, Plasmid Preparation, Dot Blot, Serial Dilution, Standard Deviation, Transfection, Imaging

    TDG activity is essential for gene reactivation. ( a ) Confocal images depicting expression levels of oxidized pOct4-mCherry expression in Tdg-/- ESCs stably rescued with GFP-TDG N151A , GFP-TDG M280H and GFP-TDG N168D in comparison to wt E14 ESCs. Scale bar: 5 μm. ( b ) High-throughput image acquisition and quantification of pOct4-mCherry expression in wt E14, Tdg-/- and Tdg-/- ESCs stably expressing wt, catalytically inactive, DNA binding deficient and caC-specific TDG mutants. Methylation of the pOct4-mCherry reporter leads to a 5-fold lower expression compared to unmodified plasmid. Oxidation of m CpG sites in the pOct4-reporter results in reactivation of mCherry-expression in wt ESCs but not in Tdg-/- ESCs. This re-increase was also obtained in Tdg-/- ESCs rescued with wt or caC-specific TDG, while the latter was not as efficient (student's t -test, ** P
    Figure Legend Snippet: TDG activity is essential for gene reactivation. ( a ) Confocal images depicting expression levels of oxidized pOct4-mCherry expression in Tdg-/- ESCs stably rescued with GFP-TDG N151A , GFP-TDG M280H and GFP-TDG N168D in comparison to wt E14 ESCs. Scale bar: 5 μm. ( b ) High-throughput image acquisition and quantification of pOct4-mCherry expression in wt E14, Tdg-/- and Tdg-/- ESCs stably expressing wt, catalytically inactive, DNA binding deficient and caC-specific TDG mutants. Methylation of the pOct4-mCherry reporter leads to a 5-fold lower expression compared to unmodified plasmid. Oxidation of m CpG sites in the pOct4-reporter results in reactivation of mCherry-expression in wt ESCs but not in Tdg-/- ESCs. This re-increase was also obtained in Tdg-/- ESCs rescued with wt or caC-specific TDG, while the latter was not as efficient (student's t -test, ** P

    Techniques Used: Activity Assay, Expressing, Stable Transfection, High Throughput Screening Assay, Binding Assay, Methylation, Plasmid Preparation

    The NEIL glycosylase family can partially compensate for TDG. ( a ) Quantification of mCherry intensities with high-throughput imaging shows that transient Tdg-/- rescue ESCs express mCherry-tagged TDG, NEIL1/2/3 and MBD4 glycosylases at comparable levels ( n = 100 000; error bars indicate standard deviation). ( b ) The ability of the NEIL family of glycosylases to substitute TDG in vivo was monitored by expression of differentially modified pOct4-GFP. With ectopic expression of mCherry-NEIL1, NEIL2 and NEIL3, reporter gene signal was significantly higher than in Tdg-/- cells, although not reaching the levels of the wt mCherry-TDG rescue. MBD4 overexpression could not compensate for loss of TDG (student's t -test, * P
    Figure Legend Snippet: The NEIL glycosylase family can partially compensate for TDG. ( a ) Quantification of mCherry intensities with high-throughput imaging shows that transient Tdg-/- rescue ESCs express mCherry-tagged TDG, NEIL1/2/3 and MBD4 glycosylases at comparable levels ( n = 100 000; error bars indicate standard deviation). ( b ) The ability of the NEIL family of glycosylases to substitute TDG in vivo was monitored by expression of differentially modified pOct4-GFP. With ectopic expression of mCherry-NEIL1, NEIL2 and NEIL3, reporter gene signal was significantly higher than in Tdg-/- cells, although not reaching the levels of the wt mCherry-TDG rescue. MBD4 overexpression could not compensate for loss of TDG (student's t -test, * P

    Techniques Used: High Throughput Screening Assay, Imaging, Standard Deviation, In Vivo, Expressing, Modification, Over Expression

    Oxidation of m CpG plasmid DNA leads to TDG-dependent gene reactivation. ( a ) Tdg-/- ESCs were transfected with pOct4-mCherry plasmids containing either unmodified, methylated or oxidized CpGs. Confocal images show a defect of ox CpG gene reactivation in Tdg-/- ESCs but not in Mbd4-/- ESCs. Transient rescue of Tdg-/- ESCs with GFP-TDG re-establishes ox CpG reporter gene expression. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. ( b ) High-throughput image acquisition and quantification of pOct4-mCherry expression shows that oxidation of m CpG sites in the pOct4-reporter results in reactivation of mCherry-expression in wt E14 ESCs and Mbd4-/- ESCs, but not in Tdg-/- ESCs. Expression of GFP-TDG rescues the phenotype (student's t -test, ** P
    Figure Legend Snippet: Oxidation of m CpG plasmid DNA leads to TDG-dependent gene reactivation. ( a ) Tdg-/- ESCs were transfected with pOct4-mCherry plasmids containing either unmodified, methylated or oxidized CpGs. Confocal images show a defect of ox CpG gene reactivation in Tdg-/- ESCs but not in Mbd4-/- ESCs. Transient rescue of Tdg-/- ESCs with GFP-TDG re-establishes ox CpG reporter gene expression. Cells were fixed with formaldehyde and counterstained with DAPI. Scale bar: 5 μm. ( b ) High-throughput image acquisition and quantification of pOct4-mCherry expression shows that oxidation of m CpG sites in the pOct4-reporter results in reactivation of mCherry-expression in wt E14 ESCs and Mbd4-/- ESCs, but not in Tdg-/- ESCs. Expression of GFP-TDG rescues the phenotype (student's t -test, ** P

    Techniques Used: Plasmid Preparation, Transfection, Methylation, Expressing, High Throughput Screening Assay

    67) Product Images from "Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation"

    Article Title: Small RNA-mediated DNA (cytosine-5) methyltransferase 1 inhibition leads to aberrant DNA methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv518

    Genome-wide DNA methylation profile of miR-155-5p transfected cells. ( A ) Scatter plot displaying differentially methylated regions in the genome after HCT116 cells were transfected with miR-155-5p or random 23-mers control RNA. The methylation difference (miR-155-5p minus random 23-mers transfected, average value of two biological replicates) was plotted against the –log 10 P- value. Green dots represent significantly changed regions. Numbers of differentially methylated regions were annotated in the plot. ( B ) Box plot showing the methylation level in replicates across the bins with increasing methylation level averages. ( C ) CpG site density curve per 10 kb in all regions, hypermethylated regions and hypomethylated regions. ( D ) Average differential methylation in genomic repetitive elements.
    Figure Legend Snippet: Genome-wide DNA methylation profile of miR-155-5p transfected cells. ( A ) Scatter plot displaying differentially methylated regions in the genome after HCT116 cells were transfected with miR-155-5p or random 23-mers control RNA. The methylation difference (miR-155-5p minus random 23-mers transfected, average value of two biological replicates) was plotted against the –log 10 P- value. Green dots represent significantly changed regions. Numbers of differentially methylated regions were annotated in the plot. ( B ) Box plot showing the methylation level in replicates across the bins with increasing methylation level averages. ( C ) CpG site density curve per 10 kb in all regions, hypermethylated regions and hypomethylated regions. ( D ) Average differential methylation in genomic repetitive elements.

    Techniques Used: Genome Wide, DNA Methylation Assay, Transfection, Methylation

    68) Product Images from "The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair"

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw274

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    69) Product Images from "Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles"

    Article Title: Indication of Horizontal DNA Gene Transfer by Extracellular Vesicles

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0163665

    Detection of A . t .-sequences in recipient cells using SYBR Green-based qPCR. (a) Standard dilutions of A . t .-DNA in duplicates with 10.000–10 copies/PCR reaction show a linear dependency whereas 1 copy/PCR was located below the detection limit. (b) Three to four replicates of DNA isolations from EV without DNase treatment (G(-); EV from harvest G without DNAse treatment) showed high abundant A . t .-sequences with Ct = 16 whereas those with DNase treatment (G(+)) showed much lower A . t .-DNA amounts with Ct near the detection limit. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 (EV from harvest C without DNase treatment, PCR run No. 16 carried out with 1μg DNA per reaction) were detected with Ct of ≥ 33. As comparison, positive standard with 10 copies/PCR was plotted. (d) Melting temperatures (Tm) of samples in (c) show the replicates with one high and several lower peaks with the correct Tm. The blue curves correspond to the positive standard of 10 copies/PCR reaction. Two exemplary arrows for sample C(-)16 in (c) and (d) point to lime and red colored probes with high and low Tm peaks, respectively.
    Figure Legend Snippet: Detection of A . t .-sequences in recipient cells using SYBR Green-based qPCR. (a) Standard dilutions of A . t .-DNA in duplicates with 10.000–10 copies/PCR reaction show a linear dependency whereas 1 copy/PCR was located below the detection limit. (b) Three to four replicates of DNA isolations from EV without DNase treatment (G(-); EV from harvest G without DNAse treatment) showed high abundant A . t .-sequences with Ct = 16 whereas those with DNase treatment (G(+)) showed much lower A . t .-DNA amounts with Ct near the detection limit. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 (EV from harvest C without DNase treatment, PCR run No. 16 carried out with 1μg DNA per reaction) were detected with Ct of ≥ 33. As comparison, positive standard with 10 copies/PCR was plotted. (d) Melting temperatures (Tm) of samples in (c) show the replicates with one high and several lower peaks with the correct Tm. The blue curves correspond to the positive standard of 10 copies/PCR reaction. Two exemplary arrows for sample C(-)16 in (c) and (d) point to lime and red colored probes with high and low Tm peaks, respectively.

    Techniques Used: SYBR Green Assay, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Detection of Venus-fluorescence and A . t .-sequences in recipient cells after passaging. (a) 2x10 5 hMSC were seeded into T25, incubated overnight to reach adherence (d0) and fed with EV derived from A . t .-hMSC cultures for 2 weeks. (b) Venus-positive cells were detected in 2 of 4 flasks (d14). One flask with 7 positive cells was passaged into 4xT25 flasks. (c) 7 days later (d21), one flask contained 10 Venus-positive cells. This culture was expanded again into 4xT25. (d) Venus-positive cells at d28 were evident in 2 flasks out of 4 with 13 cells in one flask and 1 cell in the second flask. Exemplarily, one positive MSC spot with corresponding phase-contrast for each time point is shown. Magnification x 100. (e, f) DNA of the flask with 13 Venus-positive cells was pretested in nested SYBR Green-based qPCR. Out of 10 primary reaction tubes, 4 were positive in the nested qPCR tested in 8 replicates (tubes 2, 4, 7 and 8; not shown) and were retested in TaqMan-based qPCR (e) and ddPCR (f). Shown are the results for positive control (pc, 10 copies/PCR reaction; 4 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), negative control (nc, untransduced hMSC; 8 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), and tube 2 and 4 (16 replicates in TaqMan-based qPCR and ddPCR).
    Figure Legend Snippet: Detection of Venus-fluorescence and A . t .-sequences in recipient cells after passaging. (a) 2x10 5 hMSC were seeded into T25, incubated overnight to reach adherence (d0) and fed with EV derived from A . t .-hMSC cultures for 2 weeks. (b) Venus-positive cells were detected in 2 of 4 flasks (d14). One flask with 7 positive cells was passaged into 4xT25 flasks. (c) 7 days later (d21), one flask contained 10 Venus-positive cells. This culture was expanded again into 4xT25. (d) Venus-positive cells at d28 were evident in 2 flasks out of 4 with 13 cells in one flask and 1 cell in the second flask. Exemplarily, one positive MSC spot with corresponding phase-contrast for each time point is shown. Magnification x 100. (e, f) DNA of the flask with 13 Venus-positive cells was pretested in nested SYBR Green-based qPCR. Out of 10 primary reaction tubes, 4 were positive in the nested qPCR tested in 8 replicates (tubes 2, 4, 7 and 8; not shown) and were retested in TaqMan-based qPCR (e) and ddPCR (f). Shown are the results for positive control (pc, 10 copies/PCR reaction; 4 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), negative control (nc, untransduced hMSC; 8 replicates in TaqMan-based qPCR and 2 replicates in ddPCR), and tube 2 and 4 (16 replicates in TaqMan-based qPCR and ddPCR).

    Techniques Used: Fluorescence, Passaging, Incubation, Derivative Assay, SYBR Green Assay, Real-time Polymerase Chain Reaction, Positive Control, Polymerase Chain Reaction, Negative Control

    Arabidopsis thaliana (A . t . ) virus production and transfer. (a) A . t .-DNA was cloned into the LeGO-V2-wpre plasmid vector containing Venus-fluorescence protein for detection. Primers for subsequent primary and nested A . t .-PCR shown with arrows were located within the A . t .-sequence giving rise to products of 387 bp and 106 bp respectively. (b, c) hMSC were transduced with LeGO-V2-wpre- A . t . virus supernatant. Shown is a hMSC culture 8 days after transduction (x40) detecting green cells (b) in a near confluent culture (c, phase contrast). (d, e) Recipient hMSC were incubated for 2 weeks with EV purified from hMSC- A . t . culture supernatant. Shown are Venus-positive cells (d) in the recipient culture after incubation with EV without (3 left images) or with DNase digestion (most right image) and their respective phase contrast pictures (e) (magnification x200).
    Figure Legend Snippet: Arabidopsis thaliana (A . t . ) virus production and transfer. (a) A . t .-DNA was cloned into the LeGO-V2-wpre plasmid vector containing Venus-fluorescence protein for detection. Primers for subsequent primary and nested A . t .-PCR shown with arrows were located within the A . t .-sequence giving rise to products of 387 bp and 106 bp respectively. (b, c) hMSC were transduced with LeGO-V2-wpre- A . t . virus supernatant. Shown is a hMSC culture 8 days after transduction (x40) detecting green cells (b) in a near confluent culture (c, phase contrast). (d, e) Recipient hMSC were incubated for 2 weeks with EV purified from hMSC- A . t . culture supernatant. Shown are Venus-positive cells (d) in the recipient culture after incubation with EV without (3 left images) or with DNase digestion (most right image) and their respective phase contrast pictures (e) (magnification x200).

    Techniques Used: Clone Assay, Plasmid Preparation, Fluorescence, Polymerase Chain Reaction, Sequencing, Transduction, Incubation, Purification

    Detection of A . t .-sequences in recipient cells using TaqMan-based qPCR. (a) Standard dilutions of A . t .-DNA in quadruplicates with 1.000–10 copies/PCR show a linear dependency. (b) Eightfold replicates of two different DNA isolations from EV without (G(-)) DNase treatment showed high abundant A . t .-sequences with Ct = 13 and 18 whereas those with DNase treatment (G(+)) showed much lower A . t .-DNA amounts. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 were detected with Ct of ≥ 40. As comparison, positive standard with 10 copies/PCR was plotted. All negative controls did not give rise to signals at any time (not shown).
    Figure Legend Snippet: Detection of A . t .-sequences in recipient cells using TaqMan-based qPCR. (a) Standard dilutions of A . t .-DNA in quadruplicates with 1.000–10 copies/PCR show a linear dependency. (b) Eightfold replicates of two different DNA isolations from EV without (G(-)) DNase treatment showed high abundant A . t .-sequences with Ct = 13 and 18 whereas those with DNase treatment (G(+)) showed much lower A . t .-DNA amounts. As comparison, positive standard with 100 copies/PCR was plotted. (c) Several replicates of the sample C(-)16 were detected with Ct of ≥ 40. As comparison, positive standard with 10 copies/PCR was plotted. All negative controls did not give rise to signals at any time (not shown).

    Techniques Used: Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Detection of DNA in extracellular vesicles. EV were isolated from supernatants of hMSC by ultracentrifugation, divided into two parts and DNA prepared from EV without DNase treatment (a) or after DNase treatment (b). For workflow see S4 Fig . Automatically set standards of 35 (green) and 10380 bp (pink) in the Bioanalyzer indicate the lower and upper size markers. Shown are the Bioanalyzer profiles and respective gels for a representative example. (c) Ten μl from a total of 40 μl DNA sample isolated from EV without (EV no DNase) or with (EV + DNase) DNase treatment were separated on a 0.66% agarose gel. (d) To analyze the localization, DNA was isolated from unmanipulated EV (-DNase) or EV after DNase treatment (+DNase) and examined for genomic signals in quantitative PCR using primer pairs for GAPDH, BC32-A and BD16-C1 (both randomly chosen from human genome sequences). Shown are the mean Ct values ± SD of two experiments carried out in duplicates (left graph). Products of one experiment in duplicates were visualized on 1.8% agarose gels (right blots). NTC: no template control. (e) To further elucidate the composition of EV in regard to their DNA cargo, 10 μg of protein lysate were separated on 15% SDS-PAGE and analyzed for histones H1, H2B, H3, H4 and GAPDH. As positive control, a nuclear fraction (nf) of human H1299 cells was used.
    Figure Legend Snippet: Detection of DNA in extracellular vesicles. EV were isolated from supernatants of hMSC by ultracentrifugation, divided into two parts and DNA prepared from EV without DNase treatment (a) or after DNase treatment (b). For workflow see S4 Fig . Automatically set standards of 35 (green) and 10380 bp (pink) in the Bioanalyzer indicate the lower and upper size markers. Shown are the Bioanalyzer profiles and respective gels for a representative example. (c) Ten μl from a total of 40 μl DNA sample isolated from EV without (EV no DNase) or with (EV + DNase) DNase treatment were separated on a 0.66% agarose gel. (d) To analyze the localization, DNA was isolated from unmanipulated EV (-DNase) or EV after DNase treatment (+DNase) and examined for genomic signals in quantitative PCR using primer pairs for GAPDH, BC32-A and BD16-C1 (both randomly chosen from human genome sequences). Shown are the mean Ct values ± SD of two experiments carried out in duplicates (left graph). Products of one experiment in duplicates were visualized on 1.8% agarose gels (right blots). NTC: no template control. (e) To further elucidate the composition of EV in regard to their DNA cargo, 10 μg of protein lysate were separated on 15% SDS-PAGE and analyzed for histones H1, H2B, H3, H4 and GAPDH. As positive control, a nuclear fraction (nf) of human H1299 cells was used.

    Techniques Used: Isolation, Agarose Gel Electrophoresis, Real-time Polymerase Chain Reaction, SDS Page, Positive Control

    70) Product Images from "Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response"

    Article Title: Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response

    Journal: Cell Research

    doi: 10.1038/cr.2016.118

    A functional UPR mt pathway in neurons is required for peripheral induction of UPR mt . (A) Targeting sequences of UPR mt pathway genes for CRISPR-Cas9 knockout, together with their PAM sequences. (B) Deletions of UPR mt pathway genes produced by CRISPR/Cas9 are detected by T7E1 assay. Representative DNA gels of T7E1 assay reveals the PCR products amplified from genomic DNA of control worms, or worms with UPR mt pathway gene deletion in the nervous system. (C , D) Neural knockout of atfs-1 or dve-1 suppresses the UPR mt in distal tissues in rab-3p ::Cas9+ u6p :: spg-7 -sg worms. Representative fluorescent images (C) and quantification of hsp-6p ::GFP reporter expression (D) in animals containing rab-3p ::Cas9+ u6p :: spg-7 -sg (control) or rab-3p ::Cas9+ u6p :: spg-7 -sg with neural-specific knockout of UPR mt genes are shown. mec-7p ::RFP is used as co-injection marker. n ≥ 28, error bars indicate mean ±SE. A Student's t -test is used to assess significance: * P
    Figure Legend Snippet: A functional UPR mt pathway in neurons is required for peripheral induction of UPR mt . (A) Targeting sequences of UPR mt pathway genes for CRISPR-Cas9 knockout, together with their PAM sequences. (B) Deletions of UPR mt pathway genes produced by CRISPR/Cas9 are detected by T7E1 assay. Representative DNA gels of T7E1 assay reveals the PCR products amplified from genomic DNA of control worms, or worms with UPR mt pathway gene deletion in the nervous system. (C , D) Neural knockout of atfs-1 or dve-1 suppresses the UPR mt in distal tissues in rab-3p ::Cas9+ u6p :: spg-7 -sg worms. Representative fluorescent images (C) and quantification of hsp-6p ::GFP reporter expression (D) in animals containing rab-3p ::Cas9+ u6p :: spg-7 -sg (control) or rab-3p ::Cas9+ u6p :: spg-7 -sg with neural-specific knockout of UPR mt genes are shown. mec-7p ::RFP is used as co-injection marker. n ≥ 28, error bars indicate mean ±SE. A Student's t -test is used to assess significance: * P

    Techniques Used: Functional Assay, CRISPR, Knock-Out, Produced, Polymerase Chain Reaction, Amplification, Expressing, Injection, Marker

    Neural-specific knockout of mitochondrial genes induces cell non-autonomous UPR mt . (A) Targeting sequences for CRISPR-Cas9 knockout of spg-7 or cco-1 , together with their PAM sequences. (B) Knockout of spg-7 in intestine activates hsp-6p ::GFP reporter cell autonomously. odr-1p ::dsRed is used as the co-injection marker. (C) Deletions of spg-7 or cco-1 by CRISPR/Cas9 are detected by T7E1 assay. Representative DNA gels of T7E1 assay show spg-7 or cco-1 PCR products amplified from genomic DNA of hsp-6 p::GFP worms (control), or unc-119p ::Cas9+ u6p :: spg-7 -sg(left), rab-3p ::Cas9+ u6p :: cco-1 -sg(right) worms. (D) Neural-specific knockout of spg-7 or cco-1 activates hsp-6p ::GFP in distal tissues. (E) Western blotting shows the increasing level of hsp-6p ::GFP in rab-3p ::Cas9+ u6p :: spg-7 -sg strain. The control lysate is from hsp-6p ::GFP strain. Cell lysates are probed with GFP antibody. Tubulin is used as a loading control. (F , G) Neural knockout of spg-7 fails to induce UPR ER or HSR in distal tissues. As positive controls, tunicamycin treatment robustly induces hsp-4p ::GFP reporter, heat shock 1 h at 34 °C robustly induces hsp-16.2p ::GFP reporter.
    Figure Legend Snippet: Neural-specific knockout of mitochondrial genes induces cell non-autonomous UPR mt . (A) Targeting sequences for CRISPR-Cas9 knockout of spg-7 or cco-1 , together with their PAM sequences. (B) Knockout of spg-7 in intestine activates hsp-6p ::GFP reporter cell autonomously. odr-1p ::dsRed is used as the co-injection marker. (C) Deletions of spg-7 or cco-1 by CRISPR/Cas9 are detected by T7E1 assay. Representative DNA gels of T7E1 assay show spg-7 or cco-1 PCR products amplified from genomic DNA of hsp-6 p::GFP worms (control), or unc-119p ::Cas9+ u6p :: spg-7 -sg(left), rab-3p ::Cas9+ u6p :: cco-1 -sg(right) worms. (D) Neural-specific knockout of spg-7 or cco-1 activates hsp-6p ::GFP in distal tissues. (E) Western blotting shows the increasing level of hsp-6p ::GFP in rab-3p ::Cas9+ u6p :: spg-7 -sg strain. The control lysate is from hsp-6p ::GFP strain. Cell lysates are probed with GFP antibody. Tubulin is used as a loading control. (F , G) Neural knockout of spg-7 fails to induce UPR ER or HSR in distal tissues. As positive controls, tunicamycin treatment robustly induces hsp-4p ::GFP reporter, heat shock 1 h at 34 °C robustly induces hsp-16.2p ::GFP reporter.

    Techniques Used: Knock-Out, CRISPR, Injection, Marker, Polymerase Chain Reaction, Amplification, Western Blot

    71) Product Images from "Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site"

    Article Title: Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site

    Journal: BioMed Research International

    doi: 10.1155/2016/2906484

    Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71. (a) Visual detection was performed with IMSA assay by adding HNB dye prior to amplification procedure. The color of sky blue demonstrates positive reactions while the color of violet demonstrates negative reactions. The number of the tube indicates IMSA reaction, respectively, as follows: 1: commercial Bst 2.0 DNA polymerase; 2: WT of Bst DNA pol LF; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant D540A; 7: LF mutant R412A; 8: LF mutant R412E; 9: LF mutant K416A; 10: LF mutant K416D; 11: LF mutant G310A-D540E; 12: LF mutant G310L-D540E; 13: negative control. (b) Fluorescence signals on real-time PCR instrument. Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescence detection equipment. The reaction order in (b) table was arranged the same as tubes number in (a). The sign of “+” indicates positive reactions while “−” indicates negative reactions. Reactions 1–5 were able to amplify VP1 gene to detect EV71. The curves in different colors represent distinct proteins in IMSA reaction. Curve in black and “reaction 1” represent commercial Bst 2.0 DNA polymerase. Curve in green and “reaction 2” represent WT of Bst DNA pol LF. Curve in orange and “reaction 3” represent LF mutant D540E. Curve in pink and “reaction 4” represent LF mutant G310A. Curve in red and “reaction 5” represent LF mutant G310L.
    Figure Legend Snippet: Visual IMSA assay and sensitivity evaluation of IMSA assay to test EV71. (a) Visual detection was performed with IMSA assay by adding HNB dye prior to amplification procedure. The color of sky blue demonstrates positive reactions while the color of violet demonstrates negative reactions. The number of the tube indicates IMSA reaction, respectively, as follows: 1: commercial Bst 2.0 DNA polymerase; 2: WT of Bst DNA pol LF; 3: LF mutant D540E; 4: LF mutant G310A; 5: LF mutant G310L; 6: LF mutant D540A; 7: LF mutant R412A; 8: LF mutant R412E; 9: LF mutant K416A; 10: LF mutant K416D; 11: LF mutant G310A-D540E; 12: LF mutant G310L-D540E; 13: negative control. (b) Fluorescence signals on real-time PCR instrument. Fluorescence values and curves were evaluated with Deaou-308C constant temperature fluorescence detection equipment. The reaction order in (b) table was arranged the same as tubes number in (a). The sign of “+” indicates positive reactions while “−” indicates negative reactions. Reactions 1–5 were able to amplify VP1 gene to detect EV71. The curves in different colors represent distinct proteins in IMSA reaction. Curve in black and “reaction 1” represent commercial Bst 2.0 DNA polymerase. Curve in green and “reaction 2” represent WT of Bst DNA pol LF. Curve in orange and “reaction 3” represent LF mutant D540E. Curve in pink and “reaction 4” represent LF mutant G310A. Curve in red and “reaction 5” represent LF mutant G310L.

    Techniques Used: Amplification, Mutagenesis, Negative Control, Fluorescence, Real-time Polymerase Chain Reaction

    72) Product Images from "Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis"

    Article Title: Quantitation of Murine Stroma and Selective Purification of the Human Tumor Component of Patient-Derived Xenografts for Genomic Analysis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0160587

    ssPAL analysis yields precise measurements with accuracy comparable to FACS. (A) After performing capillary electrophoresis, the presence of each PCR product (human and murine) for both primer pairs is evaluated. The peak at 206 bp corresponds to the murine fraction (orange), the peak at 211 bp correspond to the human fraction (blue). The resulting peak areas are proportional to the murine and human DNA content in a given sample. (B) ssPAL analysis is performed using two primer pairs (5 and 43) that amplify homologous regions of the mouse and human genome. This technique can accurately detect the percentages of murine DNA in pre-set mixtures of NIH 3T3 and Jurkat cells DNA within a range of 1% to 99%. Sensitivity is lost when analyzing values outside of this range. (C) FACS is the gold standard to separate human and murine cells and quantify the percentage of each population. In this representative plot, a PDX tumor from line MSK-LX29 is sorted using EpCAM and H-2K d . (D) Murine DNA content determined by ssPAL is proportional to murine cell content measured by FACS.
    Figure Legend Snippet: ssPAL analysis yields precise measurements with accuracy comparable to FACS. (A) After performing capillary electrophoresis, the presence of each PCR product (human and murine) for both primer pairs is evaluated. The peak at 206 bp corresponds to the murine fraction (orange), the peak at 211 bp correspond to the human fraction (blue). The resulting peak areas are proportional to the murine and human DNA content in a given sample. (B) ssPAL analysis is performed using two primer pairs (5 and 43) that amplify homologous regions of the mouse and human genome. This technique can accurately detect the percentages of murine DNA in pre-set mixtures of NIH 3T3 and Jurkat cells DNA within a range of 1% to 99%. Sensitivity is lost when analyzing values outside of this range. (C) FACS is the gold standard to separate human and murine cells and quantify the percentage of each population. In this representative plot, a PDX tumor from line MSK-LX29 is sorted using EpCAM and H-2K d . (D) Murine DNA content determined by ssPAL is proportional to murine cell content measured by FACS.

    Techniques Used: FACS, Electrophoresis, Polymerase Chain Reaction

    73) Product Images from "Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography"

    Article Title: Shared active site architecture between archaeal PolD and multi-subunit RNA polymerases revealed by X-ray crystallography

    Journal: Nature Communications

    doi: 10.1038/ncomms12227

    Shared active site architecture between PolD DP2 and ‘two-barrel' RNAPs. ( a ) Overview of the conserved ‘two-barrel' catalytic core in PolD DP2, S. cerevisiae RNAP-II (PDBid: 4BBS (ref. 39 )) and Neurospora crassa QDE-1 (PDBid: 2J7O (ref. 41 )). ( b ) Superposition of the DPBB subdomains of PolD (blue) and S. cerevisiae RNAP-II (pink). Left: the PolD DPBB-II subdomain is superimposed on the RNAP-II DPBB-A subdomain (Cα r.m.s.d. of 1.72 Å calculated over 73 residues). Cα of the catalytic aspartate residues are shown as spheres. Right: the PolD DPBB-I subdomain is superimposed on the RNAP-II DPBB-B subdomain (Cα r.m.s.d. of 2.21 Å calculated over 42 residues). ( c ) Possible evolutionary relationship between the DNA-dependent DNAP PolD, DNA-dependent RNAPs and RNA-dependent RNAPs. Conserved catalytic motifs are highlighted in a multi-sequence alignment. The alignment was generated using representative protein with a large sequence diversity to illustrate sequence variability (GI accession number): (i) for RNA-dependent RNAPs Caenorhabditis elegans (392,886,219), Arabidopsis thaliana (42,569,168) and N. crassa (85,091,735); (ii) for DNA-dependent RNAPs Homo sapiens (4,096,591; 119,610,588; 20,159,751), Pyrococcus abyssi (499,169,463) and Escherichia coli (983,454,941); and (iii) for D-family DNAPs P. abyssi (504,648,395), Thermococcus nautili (757,137,858), Haloferax volcanii (490,144,762), Korarchaeum cryptofilum (501,267,152) and Methanosarcina mazei (814,797,709).
    Figure Legend Snippet: Shared active site architecture between PolD DP2 and ‘two-barrel' RNAPs. ( a ) Overview of the conserved ‘two-barrel' catalytic core in PolD DP2, S. cerevisiae RNAP-II (PDBid: 4BBS (ref. 39 )) and Neurospora crassa QDE-1 (PDBid: 2J7O (ref. 41 )). ( b ) Superposition of the DPBB subdomains of PolD (blue) and S. cerevisiae RNAP-II (pink). Left: the PolD DPBB-II subdomain is superimposed on the RNAP-II DPBB-A subdomain (Cα r.m.s.d. of 1.72 Å calculated over 73 residues). Cα of the catalytic aspartate residues are shown as spheres. Right: the PolD DPBB-I subdomain is superimposed on the RNAP-II DPBB-B subdomain (Cα r.m.s.d. of 2.21 Å calculated over 42 residues). ( c ) Possible evolutionary relationship between the DNA-dependent DNAP PolD, DNA-dependent RNAPs and RNA-dependent RNAPs. Conserved catalytic motifs are highlighted in a multi-sequence alignment. The alignment was generated using representative protein with a large sequence diversity to illustrate sequence variability (GI accession number): (i) for RNA-dependent RNAPs Caenorhabditis elegans (392,886,219), Arabidopsis thaliana (42,569,168) and N. crassa (85,091,735); (ii) for DNA-dependent RNAPs Homo sapiens (4,096,591; 119,610,588; 20,159,751), Pyrococcus abyssi (499,169,463) and Escherichia coli (983,454,941); and (iii) for D-family DNAPs P. abyssi (504,648,395), Thermococcus nautili (757,137,858), Haloferax volcanii (490,144,762), Korarchaeum cryptofilum (501,267,152) and Methanosarcina mazei (814,797,709).

    Techniques Used: Sequencing, Generated

    74) Product Images from "Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans"

    Article Title: Identification and molecular characterization of bacteriophage phiAxp-2 of Achromobacter xylosoxidans

    Journal: Scientific Reports

    doi: 10.1038/srep34300

    Restriction fragment length polymorphism analysis of phiAxp-2 DNA. Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated ( HindIII ) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis of phiAxp-2 DNA. Genomic DNA from phage phiAxp-2 was digested with the enzymes indicated ( HindIII ) and run on an agarose gel (0.7%). The length of fragments generated by digestion of the linear genome or the circular genome was showed on the right side of the electrophoresis map.

    Techniques Used: Agarose Gel Electrophoresis, Generated, Electrophoresis

    75) Product Images from "Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers"

    Article Title: Identification of focally amplified lineage-specific super-enhancers in human epithelial cancers

    Journal: Nature genetics

    doi: 10.1038/ng.3470

    CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.
    Figure Legend Snippet: CRISPR/Cas9 mediated deletion of the e3 enhancer impairs the oncogenic effect of the e3 enhancer amplification (a) Upper: design of CRISPR/Cas9 mediated deletion of the e3 enhancer. Primers used to validate the e3 enhancer deletion are indicated. Bottom: Gel pictures of PCR amplification of genomic DNA using primers outside and inside the e3 enhancer region. sg-Empty: no sgRNA; sg-Control: a pair of sgRNAs that are predicted to not recognize any genomic regions; sg-e3del #1 and sg-e3del #2: two separate pairs of sgRNAs recognizing boundaries of the e3 enhancer region. PCR products were cloned into individual vectors and sequenced. Sequencing results represent the deletions induced by sg-e3del #1 (b) and sg-e3del #2 (c) . The expression level of MYC ± SEM as measured by quantitative PCR (n = 2) (d) , the cellular transformation efficiency ± SEM as measured by anchorage-independent growth (n = 3) (e) and the cellular proliferation rate ± SEM as measured by clonogenic assays (n = 3) (f) in NCI-H2009 cells with and without CRISPR/Cas9 mediated deletion of the e3 enhancer. The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (g) Schematic representation of genomic structural variants activating MYC expression in cancer.

    Techniques Used: CRISPR, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Real-time Polymerase Chain Reaction, Transformation Assay, Derivative Assay

    Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.
    Figure Legend Snippet: Identification of transcription factors required for the activity of the e3 enhancer (a) Enhancer activity ± SEM of small fragments (a-f) of the e3 enhancer as assessed by luciferase reporter assays (n = 3) in A549 LUAD cells. The fragments c and d show comparable enhancer activity relative to the intact e3 enhancer, while other fragments show minimal enhancer activity. The P -value is derived from a t-test; (***) p ≤0.001. (b) Transcription factor DNA recognition motifs are identified in the mini-e3 enhancer region that is defined by the c and d fragments overlap. (c) The luciferase reporter expression level ± SEM after deletion of individual transcription factor motif sequence in the e3 regions. The P -value is derived from a t-test (n = 3); (**) p ≤0.01; (***) p ≤0.001. (d) Luciferase reporter expression level ± SEM after silencing NFE2L2 or CEBPB by siRNA (n = 3). The P -value is derived from a t-test; (*) p ≤0.05; (**) p ≤0.01. (e) ChIP-seq profile of NFE2L2 and CEBPB in the e1–e5 enhancer regions in A549 cells.

    Techniques Used: Activity Assay, Luciferase, Derivative Assay, Expressing, Sequencing, Chromatin Immunoprecipitation

    Related Articles

    Clone Assay:

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    Article Title: Occurrence of randomly recombined functional 16S rRNA genes in Thermus thermophilus suggests genetic interoperability and promiscuity of bacterial 16S rRNAs
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    Amplification:

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    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
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    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
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    Synthesized:

    Article Title: The -11A of promoter DNA and two conserved amino acids in the melting region of ?70 both directly affect the rate limiting step in formation of the stable RNA polymerase-promoter complex, but they do not necessarily interact
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    Construct:

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
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    Electrophoresis:

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    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
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    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: For those reactions containing competitor DNA, 309 ng pUC19 DNA (New England Biolabs) was added. .. Ten percent of the reaction was removed, denatured with formamide and heated to 85 °C for 5 min. Incision products were resolved on a 10% denaturing polyacrylamide gel and electrophoresis was performed at 325 V in Tris-Borate-EDTA buffer (89 mM Tris, 89 mM boric acid and 2 mM EDTA) for 40 mins.

    Incubation:

    Article Title: Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology
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    Luciferase:

    Article Title: Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes
    Article Snippet: Restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Ipswich, MA). .. Luciferase substrate coelenterazine- h was from NanoLight Technology (Pinetop, AZ).

    Electrophoretic Mobility Shift Assay:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
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    Cell Culture:

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: The VRE strains were cultured on horse blood agar with single colonies transferred to Mueller Hinton broth (Oxoid) liquid. .. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies).

    Expressing:

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
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    BIA-KA:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: The 5’ end labeled duplex DNA containing a site specific fluorescein adducted thymine at position 26 (2 nM, F26 50/NDB) was treated with UvrABC (20 nM WT or mutant UvrA, 100 nM Bca UvrB and 50 nM Tma UvrC) in 20 µl of UvrABC buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 , 1 mM ATP and 5 mM DTT) at 55 °C for the indicated time. .. For those reactions containing competitor DNA, 309 ng pUC19 DNA (New England Biolabs) was added.

    Modification:

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    Article Title: The DnaC helicase loader is a dual ATP/ADP switch protein
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    Article Title: Occurrence of randomly recombined functional 16S rRNA genes in Thermus thermophilus suggests genetic interoperability and promiscuity of bacterial 16S rRNAs
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    Hybridization:

    Article Title: Cloning and Characterization of the Zymobacter palmae Pyruvate Decarboxylase Gene (pdc) and Comparison to Bacterial Homologues †
    Article Snippet: Restriction endonucleases and DNA-modifying enzymes were from New England BioLabs. .. Positively charged nylon membranes for Southern hybridization were from Ambion (Austin, Tex.).

    Introduce:

    Article Title: Temperature dependence of DNA persistence length
    Article Snippet: .. Obtaining mixtures of topoisomers with the equilibrium distribution of pUC19 DNA (NEB) was treated with Nt.BstNBI nicking endonuclease (NEB) to introduce single-stranded nicks. .. The nicking endonuclease was then inactivated at 65°C for 20 min. 5′-phosphates at the nicks were removed by Calf Intestinal Alkaline Phosphatase (NEB).

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
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    DNA Sequencing:

    Article Title: Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †
    Article Snippet: Restriction and DNA modification enzymes were purchased from New England Biolabs. .. DNA sequencing was carried out by the UC Berkeley DNA Sequencing Facility.

    Protein Concentration:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: Binding isotherms were fitted by nonlinear regression analysis to the equation Fb = ((1+Ka P + Ka D) - ((1 + Ka P Ka D)2 - (4DKa 2 P))1/2 )/2DT Ka ; where Fb is the fraction bound; P is the protein concentration ; DT is the total DNA concentration; Ka = 1/ Kd (app); Kd (app) is the apparent dissociation constant using Kaleidagraph and the method of Schofield [ ]. .. In those reactions containing competitor DNA, 309 ng of pUC19 DNA (New England Biolabs) was added prior to addition of the oligonucleotide.

    Sequencing:

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: .. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies). .. Enriched ISPs were loaded into ion v2 BC 316 chips (2 genomes per chip) and sequenced on an Ion PGM system (Life Technologies).

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
    Article Snippet: This construct was verified by sequencing and used for expression and purification of DNA polymerase I. .. The pUC19-VSR plasmid was constructed by digesting pUC19 DNA (New England Biolabs) with BamHI and EcoRI and inserting a duplex DNA fragment containing multiple Nt .BbvCI nicking sites.

    Recombinant:

    Article Title: Characterization of Campylobacter jejuni RacRS Reveals Roles in the Heat Shock Response, Motility, and Maintenance of Cell Length Homogeneity
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    Article Title: Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †
    Article Snippet: Paragraph title: Recombinant DNA techniques. ... Restriction and DNA modification enzymes were purchased from New England Biolabs.

    DNA Extraction:

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: Single colony cultures of these were subsequently used for DNA isolation. .. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies).

    Mutagenesis:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: The 5’ end labeled duplex DNA containing a site specific fluorescein adducted thymine at position 26 (2 nM, F26 50/NDB) was treated with UvrABC (20 nM WT or mutant UvrA, 100 nM Bca UvrB and 50 nM Tma UvrC) in 20 µl of UvrABC buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 , 1 mM ATP and 5 mM DTT) at 55 °C for the indicated time. .. For those reactions containing competitor DNA, 309 ng pUC19 DNA (New England Biolabs) was added.

    Isolation:

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: .. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies). .. Enriched ISPs were loaded into ion v2 BC 316 chips (2 genomes per chip) and sequenced on an Ion PGM system (Life Technologies).

    Article Title: Characterization of Campylobacter jejuni RacRS Reveals Roles in the Heat Shock Response, Motility, and Maintenance of Cell Length Homogeneity
    Article Snippet: DNA-modifying enzymes were purchased from New England BioLabs (Mississauga, Ontario, Canada) and Invitrogen (Burlington, Ontario, Canada). .. Plasmids were isolated from bacteria by utilizing the Qiagen Qiaprep Spin miniprep kit (Qiagen, Mississauga, Ontario, Canada).

    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
    Article Snippet: Plasmid DNA was isolated using a Nucleospin plasmid kit (Macherey & Nagel, Germany). .. Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs.

    Labeling:

    Article Title: Temperature dependence of DNA persistence length
    Article Snippet: Obtaining mixtures of topoisomers with the equilibrium distribution of pUC19 DNA (NEB) was treated with Nt.BstNBI nicking endonuclease (NEB) to introduce single-stranded nicks. .. The samples were then labeled by T4 Polynucleotide Kinase (NEB) in a 12-µl total volume, containing 7 µl of [γ-32 P]ATP [10 mCi/ml, 6000 Ci/mmol (1 Ci = 37 GBq); PerkinElmer].

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: The 5’ end labeled duplex DNA containing a site specific fluorescein adducted thymine at position 26 (2 nM, F26 50/NDB) was treated with UvrABC (20 nM WT or mutant UvrA, 100 nM Bca UvrB and 50 nM Tma UvrC) in 20 µl of UvrABC buffer (50 mM Tris-HCl pH 7.5, 50 mM KCl, 10 mM MgCl2 , 1 mM ATP and 5 mM DTT) at 55 °C for the indicated time. .. For those reactions containing competitor DNA, 309 ng pUC19 DNA (New England Biolabs) was added.

    Purification:

    Article Title: Temperature dependence of DNA persistence length
    Article Snippet: Obtaining mixtures of topoisomers with the equilibrium distribution of pUC19 DNA (NEB) was treated with Nt.BstNBI nicking endonuclease (NEB) to introduce single-stranded nicks. .. Subsequently, dephosphorylated DNA was purified from phosphatase and salts using QIAGEN PCR purification kit.

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
    Article Snippet: This construct was verified by sequencing and used for expression and purification of DNA polymerase I. .. The pUC19-VSR plasmid was constructed by digesting pUC19 DNA (New England Biolabs) with BamHI and EcoRI and inserting a duplex DNA fragment containing multiple Nt .BbvCI nicking sites.

    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
    Article Snippet: Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs. .. Purification of PCR products was performed using a Nucleospin Gel and PCR Clean-up kit (Macherey & Nagel, Germany).

    Polymerase Chain Reaction:

    Article Title: ClpAP and ClpXP degrade proteins with tags located in the interior of the primary sequence
    Article Snippet: .. Restriction endonucleases and DNA-modifying enzymes were from New England Biolabs and PCR reagents were from Perkin–Elmer Life Sciences. .. To generate pBAD-RepA-GFP a repA PCR products containing 5′ Nhe I and ribosome binding sites and a 3′ Kpn I site was cut and ligated into Nhe I- and Kpn I-digested pBAD-GFP (GFP, green fluorescent protein) ( ).

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: .. DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies). .. Enriched ISPs were loaded into ion v2 BC 316 chips (2 genomes per chip) and sequenced on an Ion PGM system (Life Technologies).

    Article Title: Temperature dependence of DNA persistence length
    Article Snippet: Obtaining mixtures of topoisomers with the equilibrium distribution of pUC19 DNA (NEB) was treated with Nt.BstNBI nicking endonuclease (NEB) to introduce single-stranded nicks. .. Subsequently, dephosphorylated DNA was purified from phosphatase and salts using QIAGEN PCR purification kit.

    Article Title: Interplay between Cyclic AMP-Cyclic AMP Receptor Protein and Cyclic di-GMP Signaling in Vibrio cholerae Biofilm Formation ▿ Biofilm Formation ▿ †
    Article Snippet: Restriction and DNA modification enzymes were purchased from New England Biolabs. .. PCRs were carried out using primers purchased from Operon Technologies (Table ) and a high-fidelity PCR kit (Roche).

    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
    Article Snippet: Oligonucleotides used for PCR and qRT-PCR were purchased from Metabion and are listed in . .. Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs.

    Article Title: Occurrence of randomly recombined functional 16S rRNA genes in Thermus thermophilus suggests genetic interoperability and promiscuity of bacterial 16S rRNAs
    Article Snippet: EmeraldAmp PCR Master Mix and In-Fusion® Cloning Kit were purchased from Takara Bio (Kusatsu, Japan). .. Restriction enzymes and DNA modification enzymes were purchased from New England Biolabs (Ipswich, MA, USA).

    Quantitative RT-PCR:

    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
    Article Snippet: Oligonucleotides used for PCR and qRT-PCR were purchased from Metabion and are listed in . .. Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs.

    Chromatin Immunoprecipitation:

    Article Title: Emergence of an Australian-like pstS-null vancomycin resistant Enterococcus faecium clone in Scotland
    Article Snippet: DNA libraries were then prepared using the NEBNext Fast DNA Fragmentation and Library Prep Set for Ion Torrent (New England Biolabs): briefly, 1 μg of genomic DNA was fragmented, and Ion Xpess barcode adapters (Life Technologies) were ligated to the DNA fragments; after clean-up using Agencourt AMPure XP beads (Beckman Coulter), 400 bp target fragments were isolated following 18 min electrophoresis on E-gel SizeSelect agarose gels (Life Technologies); these were subsequently amplified by PCR and, following another clean-up with Agencourt AMPure XP beads, the quality of the resulting DNA libraries was assessed on a 2100 Bioanalyzer (Agilent Technologies), using high sensitivity DNA chips (Agilent Technologies).Template positive Ion Sphere particles (ISPs) for semiconductor sequencing were prepared using the Ion Touch 2 System (Life Technologies). .. Enriched ISPs were loaded into ion v2 BC 316 chips (2 genomes per chip) and sequenced on an Ion PGM system (Life Technologies).

    Plasmid Preparation:

    Article Title: Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology
    Article Snippet: .. A 15-µl reaction solution containing 500 ng of plasmid DNA (pBR233, New England Biolabs) and MCM was incubated at room temperature for 30 min. About 5 U of E. coli Topoisomerase I were added to the reaction and incubated for 3 h at 37°C. .. A quantity of 25 mM EDTA and 5% SDS were added to stop the reaction which was then deproteinated by addition of proteinase K. Samples were run on a 1% agarose gel either with or without 1.4 µg/ml of the intercalator chloroquine added.

    Article Title: Reconstitution of the Very Short Patch Repair Pathway from Escherichia coli *
    Article Snippet: .. The pUC19-VSR plasmid was constructed by digesting pUC19 DNA (New England Biolabs) with BamHI and EcoRI and inserting a duplex DNA fragment containing multiple Nt .BbvCI nicking sites. ..

    Article Title: Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation
    Article Snippet: Paragraph title: DNA manipulation and plasmid construction. ... Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs.

    Binding Assay:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: Binding reactions were performed with F26 50/NDB duplex (2 nM), UvrA (20 nM) and WT UvrB (100 nM) in 20 µl of reaction buffer (50 mM Tris-HCl pH 7.5, 100 mM KCl, 10 mM MgCl2 , 1 mM ATP, 5 mM DTT and 1 µM BSA) for 15 min at 55 °C. .. In those reactions containing competitor DNA, 309 ng of pUC19 DNA (New England Biolabs) was added prior to addition of the oligonucleotide.

    Agarose Gel Electrophoresis:

    Article Title: Mini-chromosome maintenance complexes form a filament to remodel DNA structure and topology
    Article Snippet: A 15-µl reaction solution containing 500 ng of plasmid DNA (pBR233, New England Biolabs) and MCM was incubated at room temperature for 30 min. About 5 U of E. coli Topoisomerase I were added to the reaction and incubated for 3 h at 37°C. .. A quantity of 25 mM EDTA and 5% SDS were added to stop the reaction which was then deproteinated by addition of proteinase K. Samples were run on a 1% agarose gel either with or without 1.4 µg/ml of the intercalator chloroquine added.

    Produced:

    Article Title: Mutations in arrestin-3 differentially affect binding to neuropeptide Y receptor subtypes
    Article Snippet: Porcine NPY (pNPY) was produced by automated solid phase peptide synthesis using the Fmoc/tBu ( –fluorenylmethoxycarbonyl-tert-butyl) strategy, as described ( ). .. Restriction endonucleases and other DNA modifying enzymes were from New England Biolabs (Ipswich, MA).

    Concentration Assay:

    Article Title: Cooperative damage recognition by UvrA and UvrB: Identification of UvrA residues that mediate DNA binding
    Article Snippet: Binding isotherms were fitted by nonlinear regression analysis to the equation Fb = ((1+Ka P + Ka D) - ((1 + Ka P Ka D)2 - (4DKa 2 P))1/2 )/2DT Ka ; where Fb is the fraction bound; P is the protein concentration ; DT is the total DNA concentration; Ka = 1/ Kd (app); Kd (app) is the apparent dissociation constant using Kaleidagraph and the method of Schofield [ ]. .. In those reactions containing competitor DNA, 309 ng of pUC19 DNA (New England Biolabs) was added prior to addition of the oligonucleotide.

    Lysis:

    Article Title: The DnaC helicase loader is a dual ATP/ADP switch protein
    Article Snippet: Sources for reagents were as follows: radioactive nucleotides, New England Nuclear; unlabeled nucleotides, Pharmacia; ATP-γS, Sigma; and DNA modification enzymes, New England Biolabs. .. Cell lysis buffer is 20 mM Tris–HCl pH 8.0 and 100 mM NaCl.

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    TriDye 2 Log DNA Ladder 125 250 gel lanes
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    New England Biolabs indel mutation patterns dna
    Forcing CtIP recruitment to the <t>DNA</t> cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and <t>indel</t> frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P
    Indel Mutation Patterns Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs post dna transfection
    SETD1A Suppresses Fork Degradation after Replication Stress (A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post <t>transfection,</t> cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). <t>DNA</t> was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1 .
    Post Dna Transfection, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs terminal restriction fragment trf assay genomic dna
    Identification of the telomeric <t>DNA</t> sequence in A. viride . Dot blotting of A. viride genomic DNA (gDNA) using oligonucleotide ( a ) or double-stranded ( b ) telomeric probes. Control oligonucleotides and genomic DNA were included for comparison. The oligo dT 48 and bacterial genomic DNA are negative controls. ( c ) Telomeric sequences of A. viride are located at the ends of genomic DNA. Genomic DNA of A. viride was treated with Bal-31 exonuclease and then subjected to <t>TRF</t> assay using a double-stranded TTAGGG telomeric probe. In: intact genomic DNA. I: internal repetitive sequences. ( d,e ) A. viride telomeres detected by FISH in interphase nuclei ( d ) and metaphase spreads ( e ). Red fluorescent signals denote the TTAGGG telomeric sequences. Nuclei and chromosomes are in blue (DAPI). Scale bars: 10 μm.
    Terminal Restriction Fragment Trf Assay Genomic Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Forcing CtIP recruitment to the DNA cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and indel frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P

    Journal: Nature Communications

    Article Title: CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair

    doi: 10.1038/s41467-018-03475-7

    Figure Lengend Snippet: Forcing CtIP recruitment to the DNA cleavage site stimulates targeted transgene integration. a Distribution of TALEN and guide RNAs at AAVS1 safe harbor locus. gRNAs are indicated on top of their corresponding PAM motif, which is shown as lowercase in the sequence. The donor DNA used had 5′ and 3′ homology arms as indicated. b Relative HDR and indel frequencies induced by TALEN and dCas9–CtIP recruitment near the cleavage site using different guide RNAs. Human RG37 fibroblasts were transfected with the indicated plasmids and GFP transgene donor with homology arms to the targeted AAVS1 locus. HDR-mediated transgene integration was measured by FACS analysis of GFP-positive cells resulting from targeted GFP transgene integration. Indels at the cleavage site were measured by the T7E1 assay. The results are expressed as the mean of relative HDR or indel frequencies calculated by normalizing HDR or indel frequencies by that induced by TALEN transfection alone. Asterisks indicate the difference that is statistically significant when comparing cotransfection of dCas9–CtIP, guide RNA, and TALEN to TALEN-alone transfection in t -test (* P

    Article Snippet: Analysis of indel mutation patterns DNA was isolated from transfected cells (EZNA tissue DNA kit, Omega Bioteck), and the target region was amplified by PCR with Phusion Polymerase (NEB).

    Techniques: Sequencing, Transfection, FACS, Cotransfection

    SETD1A Suppresses Fork Degradation after Replication Stress (A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post transfection, cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). DNA was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1 .

    Journal: Molecular Cell

    Article Title: Histone Methylation by SETD1A Protects Nascent DNA through the Nucleosome Chaperone Activity of FANCD2

    doi: 10.1016/j.molcel.2018.05.018

    Figure Lengend Snippet: SETD1A Suppresses Fork Degradation after Replication Stress (A–F) U-2-OS cells were transfected with control siRNA or those targeting: SETD1A and SETD1B (A); BOD1L and SETD1A (B); SETD1A and SMARCAL1 (C); SETD1A and BRCA2 (D); SETD1A, BRCA2, and either KMT2C (E), or KMT2D (F). 72 hr post transfection, cells were pulsed for 20 min each with CldU and IdU and exposed to 4 mM HU for 5 hr (as in the schematic). DNA was visualized with antibodies to CldU and IdU, and tract lengths were calculated. Plots denote average ratios of IdU:CldU label length from 3 independent experiments; arrows indicate mean ratios. Plots in (E) and (F) amalgamate data from the same experiments. See also Table S1 .

    Article Snippet: 24 hr post DNA transfection, pre-existing SNAP-H3.1 was labeled with SNAP-cell 505 star (New England Biolabs) according to the manufacturer’s instructions (‘pulse’).

    Techniques: Transfection

    Identification of the telomeric DNA sequence in A. viride . Dot blotting of A. viride genomic DNA (gDNA) using oligonucleotide ( a ) or double-stranded ( b ) telomeric probes. Control oligonucleotides and genomic DNA were included for comparison. The oligo dT 48 and bacterial genomic DNA are negative controls. ( c ) Telomeric sequences of A. viride are located at the ends of genomic DNA. Genomic DNA of A. viride was treated with Bal-31 exonuclease and then subjected to TRF assay using a double-stranded TTAGGG telomeric probe. In: intact genomic DNA. I: internal repetitive sequences. ( d,e ) A. viride telomeres detected by FISH in interphase nuclei ( d ) and metaphase spreads ( e ). Red fluorescent signals denote the TTAGGG telomeric sequences. Nuclei and chromosomes are in blue (DAPI). Scale bars: 10 μm.

    Journal: Scientific Reports

    Article Title: Telomere maintenance during anterior regeneration and aging in the freshwater annelid Aeolosoma viride

    doi: 10.1038/s41598-018-36396-y

    Figure Lengend Snippet: Identification of the telomeric DNA sequence in A. viride . Dot blotting of A. viride genomic DNA (gDNA) using oligonucleotide ( a ) or double-stranded ( b ) telomeric probes. Control oligonucleotides and genomic DNA were included for comparison. The oligo dT 48 and bacterial genomic DNA are negative controls. ( c ) Telomeric sequences of A. viride are located at the ends of genomic DNA. Genomic DNA of A. viride was treated with Bal-31 exonuclease and then subjected to TRF assay using a double-stranded TTAGGG telomeric probe. In: intact genomic DNA. I: internal repetitive sequences. ( d,e ) A. viride telomeres detected by FISH in interphase nuclei ( d ) and metaphase spreads ( e ). Red fluorescent signals denote the TTAGGG telomeric sequences. Nuclei and chromosomes are in blue (DAPI). Scale bars: 10 μm.

    Article Snippet: Terminal restriction fragment (TRF) assay Genomic DNA was digested with an Rsa I and Hinf I (NEB) endonuclease mixture (1:1) at 37 °C overnight and then resolved in a 1% agarose gel.

    Techniques: Sequencing, TRF Assay, Fluorescence In Situ Hybridization