λ exonuclease buffer  (New England Biolabs)


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    Structured Review

    New England Biolabs λ exonuclease buffer
    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and <t>λ-exonuclease</t> treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.
    λ Exonuclease Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection"

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00166-17

    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.
    Figure Legend Snippet: Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Techniques Used: In Situ, Infection, Plasmid Purification

    2) Product Images from "Lamins organize the global three-dimensional genome from the nuclear periphery"

    Article Title: Lamins organize the global three-dimensional genome from the nuclear periphery

    Journal: bioRxiv

    doi: 10.1101/211656

    Expansion of HiLands-P upon lamin loss A. A plot of log 2 fold increased or decreased total interactions between two HiLands-P regions upon lamin loss as a function of the distance between the regions. B. FISH probe production. PCR1 amplifies the probes for a specific sub-library using the indicated sub-library primers. PCR2 produces the labeled sub-library probes using the fluorescently labeled and phosphorylated common primers. Lambda exonuclease digests the phosphorylated DNA strand to produce the single stranded DNA probes for FISH. C. Four regions (dashed boxes) on Chromosome 1, 4, 13, and 14 consisting of mostly HiLands-P were selected for FISH. HiLands are shown in corresponding colors. D. Box plot showing the log 2 fold change of inter-TAD interactions for 20-Kb windows in the whole genome (All) or in selected chromosome regions shown in C. Only HiLands-P interactions are included. E. Two representative 3D-projection FISH images for each of the four selected regions in C. Purple: DAPI staining for DNA. White: FISH signal. The white dashed lines demarcate the boundaries of nuclei that are next to one another. Scale bars, 5 µm. The volume and surface areas of the four chromatin regions are quantified to the right. P-values, Wilcoxon rank-sum test.
    Figure Legend Snippet: Expansion of HiLands-P upon lamin loss A. A plot of log 2 fold increased or decreased total interactions between two HiLands-P regions upon lamin loss as a function of the distance between the regions. B. FISH probe production. PCR1 amplifies the probes for a specific sub-library using the indicated sub-library primers. PCR2 produces the labeled sub-library probes using the fluorescently labeled and phosphorylated common primers. Lambda exonuclease digests the phosphorylated DNA strand to produce the single stranded DNA probes for FISH. C. Four regions (dashed boxes) on Chromosome 1, 4, 13, and 14 consisting of mostly HiLands-P were selected for FISH. HiLands are shown in corresponding colors. D. Box plot showing the log 2 fold change of inter-TAD interactions for 20-Kb windows in the whole genome (All) or in selected chromosome regions shown in C. Only HiLands-P interactions are included. E. Two representative 3D-projection FISH images for each of the four selected regions in C. Purple: DAPI staining for DNA. White: FISH signal. The white dashed lines demarcate the boundaries of nuclei that are next to one another. Scale bars, 5 µm. The volume and surface areas of the four chromatin regions are quantified to the right. P-values, Wilcoxon rank-sum test.

    Techniques Used: Fluorescence In Situ Hybridization, Labeling, Staining

    3) Product Images from "A low-bias and sensitive small RNA library preparation method using randomized splint ligation"

    Article Title: A low-bias and sensitive small RNA library preparation method using randomized splint ligation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa480

    Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.
    Figure Legend Snippet: Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

    Techniques Used: Ligation, Synthesized, Polymerase Chain Reaction

    4) 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

    MRN can stimulate resection by an Mre11 nuclease-independent mechanism. ( A ) Effect of MRN on the activities of lambda exonuclease and Exo1 against 3′ avidin DNA. ( B ) Comparison of the wild-type and mutant MRN complexes on the stimulation of Exo1 activity against 3′ avidin DNA. ( C ) Plot of the amounts of 3′ 32 P on the remaining substrates at the indicated times. The averages and standard deviations were calculated with three sets of data.
    Figure Legend Snippet: MRN can stimulate resection by an Mre11 nuclease-independent mechanism. ( A ) Effect of MRN on the activities of lambda exonuclease and Exo1 against 3′ avidin DNA. ( B ) Comparison of the wild-type and mutant MRN complexes on the stimulation of Exo1 activity against 3′ avidin DNA. ( C ) Plot of the amounts of 3′ 32 P on the remaining substrates at the indicated times. The averages and standard deviations were calculated with three sets of data.

    Techniques Used: Avidin-Biotin Assay, Mutagenesis, Activity Assay

    5) Product Images from "First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid"

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029884

    PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.
    Figure Legend Snippet: PFGE and Southern blot hybridization with IS 1245 -derived probe of M. avium and M. kansasii colonies. (A) PFGE with undigested DNA; (B) Southern blot hybridization with IS 1245 -derived probe. Open arrow indicates pMA100; closed arrow indicates the uncharacterized smaller hybridization band. 88.1 to 88.4 = M . avium ; 88.5 to 88.75 = PCR−IS 1245- negative M . kansasii ; 88.8 to 88.15 = PCR−IS 1245- positive M. kansasii. On the left, Lambda Ladder PFG Marker (NewEngland BioLabs) molecular size markers.

    Techniques Used: Southern Blot, Hybridization, Derivative Assay, Polymerase Chain Reaction, Marker

    PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.
    Figure Legend Snippet: PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Techniques Used: Lysis

    6) Product Images from "Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template"

    Article Title: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template

    Journal: Synthetic Biology

    doi: 10.1093/synbio/ysaa030

    Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.
    Figure Legend Snippet: Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.

    Techniques Used: Amplification, Introduce, Ligation, Polymerase Chain Reaction

    7) Product Images from "ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints"

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3121

    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.
    Figure Legend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.
    Figure Legend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Variant Assay, Binding Assay

    8) Product Images from "Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection"

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    Journal: Journal of Virology

    doi: 10.1128/JVI.00166-17

    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.
    Figure Legend Snippet: Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Techniques Used: In Situ, Infection, Plasmid Purification

    9) Product Images from "DSBCapture: in situ capture and direct sequencing of dsDNA breaks"

    Article Title: DSBCapture: in situ capture and direct sequencing of dsDNA breaks

    Journal: Nature methods

    doi: 10.1038/nmeth.3960

    DSBCapture methodology and validation ( a ) DSBCapture workflow. (1) DSBs in fixed nuclei were blunt-end repaired, (2) A-tailed and (3) ligated to a biotinylated (black ball) modified P5 Illumina adapter (orange lines). (4) Excess adapters were removed by lambda exonuclease digestion; (5) DNA extracted from lysed nuclei was fragmented by sonication, (6) bead-captured (hollow ball) and blunted-end repaired, (7) A-tailed, and (8) ligated to a modified P7 Illumina adapter (purple lines). (9) Captured break sites were PCR amplified, (10) size selected, (11) quantified and (12) sequenced. Sequences of the DSBCapture adapters: modified P5 Illumina adapter and modified P7 Illumina adapter (B = biotin; P = phosphorylated; * = phosphorothioate bond). ( b ) DSBs created by EcoRV cleavage in fixed nuclei (N = 1). PCR duplicates have been removed. Data range is shown in square brackets and black boxes illustrate the genomic location of EcoRV sites. A 20 kb region and a 110 bp region are shown. Pink and purple lines: reads from the sense and antisense strand, respectively. As EcoRV is a blunt cutter, reads originate directly from the cleavage site. ( c ) AsiSI cleavage sites (black boxes) detected by DSBCapture (N = 1). Cleavage by AsiSI generates a 2 bp 3’ overhang; end processing removes this overhang generating the 2 bp gap in the center of the peak. A 2 kb and a 200 bp region are shown. ( d ) Venn diagram illustrating the overlap of DSBs detected at AsiSI sites by DSBCapture and γH2AX ChIP-seq 9 .
    Figure Legend Snippet: DSBCapture methodology and validation ( a ) DSBCapture workflow. (1) DSBs in fixed nuclei were blunt-end repaired, (2) A-tailed and (3) ligated to a biotinylated (black ball) modified P5 Illumina adapter (orange lines). (4) Excess adapters were removed by lambda exonuclease digestion; (5) DNA extracted from lysed nuclei was fragmented by sonication, (6) bead-captured (hollow ball) and blunted-end repaired, (7) A-tailed, and (8) ligated to a modified P7 Illumina adapter (purple lines). (9) Captured break sites were PCR amplified, (10) size selected, (11) quantified and (12) sequenced. Sequences of the DSBCapture adapters: modified P5 Illumina adapter and modified P7 Illumina adapter (B = biotin; P = phosphorylated; * = phosphorothioate bond). ( b ) DSBs created by EcoRV cleavage in fixed nuclei (N = 1). PCR duplicates have been removed. Data range is shown in square brackets and black boxes illustrate the genomic location of EcoRV sites. A 20 kb region and a 110 bp region are shown. Pink and purple lines: reads from the sense and antisense strand, respectively. As EcoRV is a blunt cutter, reads originate directly from the cleavage site. ( c ) AsiSI cleavage sites (black boxes) detected by DSBCapture (N = 1). Cleavage by AsiSI generates a 2 bp 3’ overhang; end processing removes this overhang generating the 2 bp gap in the center of the peak. A 2 kb and a 200 bp region are shown. ( d ) Venn diagram illustrating the overlap of DSBs detected at AsiSI sites by DSBCapture and γH2AX ChIP-seq 9 .

    Techniques Used: Modification, Sonication, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation

    10) Product Images from "Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries"

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0094752

    Exonucleolytic hydrolysis of 5 ′ -phosphorylated strand. (A) Experimental design. (B) lane A – emulsion PCR product (step 2), lane B – exonuclease I hydrolysis of PCR product, lane C – ssDNA product of the lambda exonuclease treatment after removal of PBS (fig. 4 step 6; c), and lane D – exonuclease I hydrolysis of PBS-free ssDNA.
    Figure Legend Snippet: Exonucleolytic hydrolysis of 5 ′ -phosphorylated strand. (A) Experimental design. (B) lane A – emulsion PCR product (step 2), lane B – exonuclease I hydrolysis of PCR product, lane C – ssDNA product of the lambda exonuclease treatment after removal of PBS (fig. 4 step 6; c), and lane D – exonuclease I hydrolysis of PBS-free ssDNA.

    Techniques Used: Polymerase Chain Reaction

    11) Product Images from "Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction"

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky154

    The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).
    Figure Legend Snippet: The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).

    Techniques Used: Fluorescence, Standard Deviation, Sequencing, Labeling, Agarose Gel Electrophoresis

    ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.
    Figure Legend Snippet: ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.

    Techniques Used:

    Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.
    Figure Legend Snippet: Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.

    Techniques Used:

    12) Product Images from "An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation"

    Article Title: An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation

    Journal: Cells

    doi: 10.3390/cells10051076

    Schematic diagram of knock-in mouse generation using long ssDNA donors prepared by the phospho-PCR method. ( A ) Flowchart of the phospho-PCR method. A dsDNA template containing a gene cassette of interest (GOI) flanked by homology arms on both sides is depicted at the top. A pair of normal primer and 5′-phospholyrated primer for PCR is used to amplify the starting dsDNA substrates for exonuclease reactions. Strandase Mix A or Lambda exonuclease selectively degrades the phosphorylated chains of the duplex. Strandase Mix B or Exonuclease III subsequently help complete the selective degradations to yield long ssDNAs. Minor PCR products without 5′-phosphorylations are also extensively digested by Exonuclease III activities, whose efficiency is detailed in Results Section 3.2 . Note that this sequential use of two exonucleases enables highly pure ssDNA production. ( B ) Schematic of CRISPR/Cas9 mediated knock-in mouse generation using long ssDNA donors. Long ssDNA donor and Cas9-guide RNA complex can be introduced into mouse fertilized eggs either by pronuclear injection or by electroporation. Treated eggs are transferred into the oviduct of pseudo-pregnant recipients to obtain knock-in founders.
    Figure Legend Snippet: Schematic diagram of knock-in mouse generation using long ssDNA donors prepared by the phospho-PCR method. ( A ) Flowchart of the phospho-PCR method. A dsDNA template containing a gene cassette of interest (GOI) flanked by homology arms on both sides is depicted at the top. A pair of normal primer and 5′-phospholyrated primer for PCR is used to amplify the starting dsDNA substrates for exonuclease reactions. Strandase Mix A or Lambda exonuclease selectively degrades the phosphorylated chains of the duplex. Strandase Mix B or Exonuclease III subsequently help complete the selective degradations to yield long ssDNAs. Minor PCR products without 5′-phosphorylations are also extensively digested by Exonuclease III activities, whose efficiency is detailed in Results Section 3.2 . Note that this sequential use of two exonucleases enables highly pure ssDNA production. ( B ) Schematic of CRISPR/Cas9 mediated knock-in mouse generation using long ssDNA donors. Long ssDNA donor and Cas9-guide RNA complex can be introduced into mouse fertilized eggs either by pronuclear injection or by electroporation. Treated eggs are transferred into the oviduct of pseudo-pregnant recipients to obtain knock-in founders.

    Techniques Used: Knock-In, Polymerase Chain Reaction, CRISPR, Injection, Electroporation

    Purely amplified PCR products and the following sequential use of two exonucleases enabled high-quality ssDNA production. ( A ) Optimization of PCR conditions to obtain a clear single product was the first key step for phospho-PCR-mediated ssDNA production. Primers’ concentrations, annealing temperature, and templates’ concentration for the PCR reaction depicted in Figure 2 B were optimized. The manufacturer’s protocols are underlined, and our optimized conditions are colored in blue. To obtain a single product with high efficiency, the primers’ concentrations should be lower, the annealing temperature should be higher, and the templates’ concentration should be much lower than the manufacturer’s instructions. Asterisks (*) indicate the unwanted non-specific amplifications. Blue triangle indicates the purely amplified products. ( B ) Sequential use of two exonucleases depicted in Figure 2 B allowed for highly pure long ssDNA production. While the single use of Strandase Mix A or Lambda exonuclease selectively degraded 5′-phosphorylated strands to yield ssDNAs (red triangles), dsDNA substrates indicated by asterisks remained in the reaction mixtures. Subsequently added Strandase Mix B or Exonuclease III degraded the remaining dsDNAs to yield pure long ssDNAs. Note that the long ssDNA treated with Exonuclease III was slightly shorter than that treated with Strandase Mix B.
    Figure Legend Snippet: Purely amplified PCR products and the following sequential use of two exonucleases enabled high-quality ssDNA production. ( A ) Optimization of PCR conditions to obtain a clear single product was the first key step for phospho-PCR-mediated ssDNA production. Primers’ concentrations, annealing temperature, and templates’ concentration for the PCR reaction depicted in Figure 2 B were optimized. The manufacturer’s protocols are underlined, and our optimized conditions are colored in blue. To obtain a single product with high efficiency, the primers’ concentrations should be lower, the annealing temperature should be higher, and the templates’ concentration should be much lower than the manufacturer’s instructions. Asterisks (*) indicate the unwanted non-specific amplifications. Blue triangle indicates the purely amplified products. ( B ) Sequential use of two exonucleases depicted in Figure 2 B allowed for highly pure long ssDNA production. While the single use of Strandase Mix A or Lambda exonuclease selectively degraded 5′-phosphorylated strands to yield ssDNAs (red triangles), dsDNA substrates indicated by asterisks remained in the reaction mixtures. Subsequently added Strandase Mix B or Exonuclease III degraded the remaining dsDNAs to yield pure long ssDNAs. Note that the long ssDNA treated with Exonuclease III was slightly shorter than that treated with Strandase Mix B.

    Techniques Used: Amplification, Polymerase Chain Reaction, Concentration Assay

    Mouse Dcx locus as a model site for targeted recombinase knock-in. ( A ) Targeting strategy to insert an iCre recombinase cassette just upstream from the translational stop codon in the Dcx gene is outlined. Schematic of the Dcx gene structure, the guide RNA sequences, and the long ssDNA donor is shown in the upper half. The resulting knock-in allele and its genotyping primers are depicted in the lower half. T2A peptide sequences were employed for bicistronic iCre expressions in Dcx-expressing cells. ( B ) The flowchart of long ssDNA production for Dcx-T2A-iCre knock-in via the phospho-PCR method is depicted. The artificially synthesized gene containing a T2A-iCre cassette was used as the template for phospho-PCR. 5′-phosphorylated strands of the PCR products were sequentially digested by Lambda exonuclease and Exonuclease III to yield long ssDNA donor. ( C ) PCR screening results for knock-in newborns derived from the pronuclear injection shown in Table 2 (Dcx-T2A-iCre) are summarized. Two pairs of primers depicted in ( A ) were used to confirm the designed knock-in. Dcx_Fw and Dcx_Rv were designed outside from the donor DNA’s homology arms to exclude the detection of unintended random integrations. Asterisks indicate the knock-in founders with correct insertions (No.3, No.5, No.6, and No.8). No.5 carried an additional incorrect knock-in allele. ( D ) Boundary sequences between the Dcx gene and T2A-iCre cassette analyzed by using the genome DNA from No.3 founder in ( C ) are aligned.
    Figure Legend Snippet: Mouse Dcx locus as a model site for targeted recombinase knock-in. ( A ) Targeting strategy to insert an iCre recombinase cassette just upstream from the translational stop codon in the Dcx gene is outlined. Schematic of the Dcx gene structure, the guide RNA sequences, and the long ssDNA donor is shown in the upper half. The resulting knock-in allele and its genotyping primers are depicted in the lower half. T2A peptide sequences were employed for bicistronic iCre expressions in Dcx-expressing cells. ( B ) The flowchart of long ssDNA production for Dcx-T2A-iCre knock-in via the phospho-PCR method is depicted. The artificially synthesized gene containing a T2A-iCre cassette was used as the template for phospho-PCR. 5′-phosphorylated strands of the PCR products were sequentially digested by Lambda exonuclease and Exonuclease III to yield long ssDNA donor. ( C ) PCR screening results for knock-in newborns derived from the pronuclear injection shown in Table 2 (Dcx-T2A-iCre) are summarized. Two pairs of primers depicted in ( A ) were used to confirm the designed knock-in. Dcx_Fw and Dcx_Rv were designed outside from the donor DNA’s homology arms to exclude the detection of unintended random integrations. Asterisks indicate the knock-in founders with correct insertions (No.3, No.5, No.6, and No.8). No.5 carried an additional incorrect knock-in allele. ( D ) Boundary sequences between the Dcx gene and T2A-iCre cassette analyzed by using the genome DNA from No.3 founder in ( C ) are aligned.

    Techniques Used: Knock-In, Expressing, Polymerase Chain Reaction, Synthesized, Derivative Assay, Injection

    13) Product Images from "Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae"

    Article Title: Functional characterization of an alkaline exonuclease and single strand annealing protein from the SXT genetic element of Vibrio cholerae

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-12-16

    Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo . In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl 2 . Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D : Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.
    Figure Legend Snippet: Digestion of fluorescently-labeled annealed oligonucleotide substrates by lambda-Exo . In experiments analogous to those described for SXT-Exo (see Figure 7), the ability of lambda-Exo to digest three different (partially) dsDNA substrates was investigated. In each assay, lambda-Exo (3 pmol of trimers) was incubated at 25°C with 20 pmol of the dsDNA substrate in 50 mM Tris-HCl pH8.0, 5 mM MgCl 2 . Aliquots were removed and quenched at 0, 0.5, 1, 2, 4 and 10 minutes; then analyzed on 7 M urea-TBE denaturing polyacrylamide gels (times indicated above lanes). Gels were scanned for fluorescence, and fluorescence intensities of the bands corresponding to the Cy3-labeled strand were quantified. Panel A : Representative fluorescence-scanned gel image showing time-wise digestion of the 5'-overhang DNA substrate by SXT-Exo. Panel B : Representative gel image showing digestion of the Blunt ended DNA substrate by lambda-Exo. Panel C : Representative gel image showing digestion of the 3'-overhang DNA substrate by lambda-Exo. Panel D : Plot showing the digestion of the three DNA substrates by lambda-Exo over a 10 minute period; reported as the mean percentage ± standard deviation, based on three independent replicates. See materials section for details.

    Techniques Used: Labeling, Incubation, Fluorescence, Standard Deviation

    Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).
    Figure Legend Snippet: Qualitative analysis of the metal ion dependence, DNA substrate preferences and mode of digestion of the SXT-Exo alkaline exonuclease . Panel A : Agarose gel showing ability of SXT-Exo to digest linear dsDNA (NdeI-linerized pET28a; lanes 2-5), circularized dsDNA (undigested pET28a; lanes 6 and 7), circularized ssDNA (M13 phage DNA; lanes 8 and 9) in Tris-HCl pH7.4, 50 mM NaCl with/without 10 mM MgCl 2 ; λ-HindIII (NEB) DNA ladder (lane1). Panel B : Agarose gel showing the ability of SXT-Exo and lambda-Exo to digest 5'-phosphorylated linear dsDNA substrates ('unmodified'; lanes 2, 3, 6 and 7), compared with analogous 5'-phosphorylated linear dsDNA substrates containing 3 consecutive phosphorothioate linkages at the 5'-termini of each strand (PT-modified; lanes 4, 5, 8 and 9). The 712 bp 'unmodified' or 'PT-modified' dsDNA substrates (0.1 mg) were incubated at 37°C with lambda-Exo (3 μg) or SXT-Exo (30 μg) in Tris-HCl, (25 mM, pH7.4), 50 mM NaCl, 10 mM MgCl 2 (total volume 40 μl). Aliquots (20 μl) were quenched (20 mM EDTA + 1% SDS) immediately, and after 30 mins, and analyzed on 1% agarose TAE gels. 1 Kb Plus DNA Ladder (Invitrogen; lane 1). Panel C : Agarose gel showing time-course of digestion of 5'-phosphorylated linear dsDNA (NdeI-linearized pET28a, 0.56 pmol) by SXT-Exo (50 pmol of trimers) in Tris-HCl pH7.4, 50 mM NaCl, 10 mM MgCl 2 ; at 37°C, with aliquots removed at times indicated (0-160 minutes; lanes 2-11); 1 Kb Plus DNA Ladder (lane 1).

    Techniques Used: Agarose Gel Electrophoresis, Modification, Incubation

    Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.
    Figure Legend Snippet: Purification of SXT-Exo and lambda-Exo, and determination of their multimericity by size exclusion chromatography . Panel A : Size exclusion chromatogram of purified SXT-Exo protein expressed from plasmid pEA1-1. Panel B : Size exclusion chromatogram of purified lambda-Exo protein expressed from plasmid pEE4. Panel C : 12% polyacrylamide gel (SDS-PAGE) analysis of the SXT-Exo purification procedure and purified SXT-Bet, SXT-Ssb, lambda-Bet and lambda-Exo proteins; lane 1: Benchmark protein ladder (Invitrogen); lane 2: pEA1-1/ E. coli BL21 (DE3) pLysS Rosetta whole cell extract immediately prior to induction; lane 3: whole cell extract 6 hours after induction with IPTG; lane 4: supernatant from cell extract 6 hours post induction; lane 5: purified SXT-Exo; lane 6: purified SXT-Bet expressed from pX28-1; lane 7: purified SXT-Ssb expressed from pSB2; lane 8: purified lambda-Bet expressed from p1DB; lane 9: purified lambda-Exo expressed from pEE4.

    Techniques Used: Purification, Size-exclusion Chromatography, Plasmid Preparation, SDS Page

    Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P
    Figure Legend Snippet: Stimulation of double strand DNA exonuclease activities of SXT-Exo and lambda-Exo by SSAP and Ssb proteins . Panel A . SXT-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of the protein indicated in the text (BSA, lambda-Bet, SXT-Bet or SXT-Ssb) in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 0.5 mM MnCl 2 ; were incubated at 25°C for 30 mins before EDTA quenching. dsDNA levels were immediately quantified using PicoGreen reagent. The level of DNA digestion by SXT-Exo in the absence of added protein (-) was normalized to a value of 100%. Panel B . In analogous sets of experiments, lambda-Exo (2 pmol of trimers), PstI-linearized pUC18 (5 ng, 0.003 pmol) and 2 pmol of BSA, lambda-Bet, SXT-Bet or SXT-Ssb; in Tris-HCl (25 mM, pH7.4), 50 mM NaCl, 5 mM MgCl 2 ; were incubated at 25°C for 10 mins. Digestion levels were normalized to those of lambda-Exo in the absence of added protein (-). See methods section for detailed experimental procedure. Six independent replicates were performed for each experiment, and error bars indicate standard deviation from the mean values. Analysis using ANOVA indicated all results were statistically significant (P

    Techniques Used: Incubation, Standard Deviation

    14) Product Images from "Transcription-generated torsional stress destabilizes nucleosomes"

    Article Title: Transcription-generated torsional stress destabilizes nucleosomes

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2723

    High resolution detection of supercoiling states ( a ) Strategy for paired-end sequencing of TMP cross-linked DNA fragments. I. Illumina barcoded adapters are ligated to cross-linked fragments. II. The 5′ strand is digested with λ exonuclease. III. Using a primer complementary to the paired-end adapter, 10 rounds of primer extension were performed. IV. Ribo-Gs were added at the 3′ end using Terminal Transferase, V. A double stranded adapter with a 5′ CCC overhang was ligated. VI. One round of primer extension followed by cycles of library amplification were performed. Libraries were sequenced from the CCC overhang end. ( b ) TMP-seq was performed on control samples with two replicates. Reads were normalized for DNA sequence bias. The average normalized TMP-seq (y-axis Ave. sample/DNA) signal for every 10 bp in a 4 kb region surrounding the transcription start site (TSS) and transcription end site (TES) of all genes is plotted (top), and for expressed and silent genes separately (bottom). n.c. Normalized counts.
    Figure Legend Snippet: High resolution detection of supercoiling states ( a ) Strategy for paired-end sequencing of TMP cross-linked DNA fragments. I. Illumina barcoded adapters are ligated to cross-linked fragments. II. The 5′ strand is digested with λ exonuclease. III. Using a primer complementary to the paired-end adapter, 10 rounds of primer extension were performed. IV. Ribo-Gs were added at the 3′ end using Terminal Transferase, V. A double stranded adapter with a 5′ CCC overhang was ligated. VI. One round of primer extension followed by cycles of library amplification were performed. Libraries were sequenced from the CCC overhang end. ( b ) TMP-seq was performed on control samples with two replicates. Reads were normalized for DNA sequence bias. The average normalized TMP-seq (y-axis Ave. sample/DNA) signal for every 10 bp in a 4 kb region surrounding the transcription start site (TSS) and transcription end site (TES) of all genes is plotted (top), and for expressed and silent genes separately (bottom). n.c. Normalized counts.

    Techniques Used: Sequencing, Countercurrent Chromatography, Amplification

    15) Product Images from "High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing"

    Article Title: High Precision Detection of Rare Splice Isoforms Using Multiplexed Primer Extension Sequencing

    Journal: bioRxiv

    doi: 10.1101/331629

    Array-based oligonucleotide synthesis can be used to generate primer pools for use in MPE-seq (A) Obtaining adequate amounts of primer pools for MPE-seq from cost-effective array-based oligonucleotide synthesis can be achieved in four steps. (1) PCR amplification of the oligonucleotide synthesis pool using a 5’ blocked sense primer and a biotinylated antisense primer. (2) Restriction digestion to cleave off the PCR primer handle. (3) Lambda exonuclease digestion of free 5’ ends. (4) Streptavidin purification of biotinylated PCR handle. The unbound fraction is the desired primer pool product. (B) A scatter plot compares the fraction of unspliced mRNA measured by MPE-seq libraries which used individually synthesized primer pools versus array-based synthesis of primer pools. (C) The percentage of reads mapped to target and off-target regions is depicted for MPE-seq using array-synthesized primers. (D) Sashimi plot of a targeted region within the ats1 gene locus demonstrates the capacity of MPE-seq to reveal complex alternative splicing patterns with higher sensitivity than RNA-seq, despite having lower total sequencing depth.
    Figure Legend Snippet: Array-based oligonucleotide synthesis can be used to generate primer pools for use in MPE-seq (A) Obtaining adequate amounts of primer pools for MPE-seq from cost-effective array-based oligonucleotide synthesis can be achieved in four steps. (1) PCR amplification of the oligonucleotide synthesis pool using a 5’ blocked sense primer and a biotinylated antisense primer. (2) Restriction digestion to cleave off the PCR primer handle. (3) Lambda exonuclease digestion of free 5’ ends. (4) Streptavidin purification of biotinylated PCR handle. The unbound fraction is the desired primer pool product. (B) A scatter plot compares the fraction of unspliced mRNA measured by MPE-seq libraries which used individually synthesized primer pools versus array-based synthesis of primer pools. (C) The percentage of reads mapped to target and off-target regions is depicted for MPE-seq using array-synthesized primers. (D) Sashimi plot of a targeted region within the ats1 gene locus demonstrates the capacity of MPE-seq to reveal complex alternative splicing patterns with higher sensitivity than RNA-seq, despite having lower total sequencing depth.

    Techniques Used: Oligonucleotide Synthesis, Polymerase Chain Reaction, Amplification, Purification, Synthesized, RNA Sequencing Assay, Sequencing

    Array-based oligonucleotide synthesis can be used to generate primer pools for use in MPE-seq Steps during the amplification and purification of array-synthesized primer pools are monitored via native gel electrophoresis. The control lane represents a pool of individually synthesized MPE-seq primers which did not require amplification and purification. Lanes refer to products of each individual step in the protocol. (1) PCR amplification of the oligonucleotide synthesis pool using a 5’ blocked sense primer and a biotinylated antisense primer. (2) Restriction digestion to cleave off the PCR primer handle. (3) Lambda exonuclease digestion of free 5’ ends. (4) Streptavidin purification of biotinylated PCR handle. The unbound fraction is the desired primer pool product.
    Figure Legend Snippet: Array-based oligonucleotide synthesis can be used to generate primer pools for use in MPE-seq Steps during the amplification and purification of array-synthesized primer pools are monitored via native gel electrophoresis. The control lane represents a pool of individually synthesized MPE-seq primers which did not require amplification and purification. Lanes refer to products of each individual step in the protocol. (1) PCR amplification of the oligonucleotide synthesis pool using a 5’ blocked sense primer and a biotinylated antisense primer. (2) Restriction digestion to cleave off the PCR primer handle. (3) Lambda exonuclease digestion of free 5’ ends. (4) Streptavidin purification of biotinylated PCR handle. The unbound fraction is the desired primer pool product.

    Techniques Used: Oligonucleotide Synthesis, Amplification, Purification, Synthesized, Nucleic Acid Electrophoresis, Polymerase Chain Reaction

    16) Product Images from "Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction"

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky154

    The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).
    Figure Legend Snippet: The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).

    Techniques Used: Fluorescence, Standard Deviation, Sequencing, Labeling, Agarose Gel Electrophoresis

    ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.
    Figure Legend Snippet: ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.

    Techniques Used:

    Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.
    Figure Legend Snippet: Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.

    Techniques Used:

    17) Product Images from "Single cell epigenetic visualization assay"

    Article Title: Single cell epigenetic visualization assay

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab009

    HIV provirus in latently infected cells. ( A ) Schematic overview of the HIV genome and the EVA probe (green bar) that covers 5′ 5kb region of the HIV-1 genome. In the used cell lines (J-Lat), the ENV gene is mutated (red cross), and the NEF gene is replaced with GFP (green box). ( B ) Representative EVA images of the HIV locus in J-Lat 8.4 cell line that contains one copy of the HIV-1 provirus. EVA was done with 5mC antibody (upper row) or without antibody (lower row). Arrows indicate positions of red foci. Scale bar, 5 μm. ( C ) Quantitation of DNA methylation levels of the HIV-1 proviral genome and dynamic range of the HIV EVA signal measurements. Orange: green-to-red EVA signal ratios of HIV locus with (5mC Ab)and without 5mC antibody (no Ab). Blue: HIV EVA was done in the same cells without lambda exonuclease treatment to estimate maximum green signal intensity and technical noise. Different proportions of green oligo were mixed with the same unlabeled oligo (0.00, 0.33, 0.67, 1.00 labeled-to-unlabeled oligo ratios). Each dot represents one cell.
    Figure Legend Snippet: HIV provirus in latently infected cells. ( A ) Schematic overview of the HIV genome and the EVA probe (green bar) that covers 5′ 5kb region of the HIV-1 genome. In the used cell lines (J-Lat), the ENV gene is mutated (red cross), and the NEF gene is replaced with GFP (green box). ( B ) Representative EVA images of the HIV locus in J-Lat 8.4 cell line that contains one copy of the HIV-1 provirus. EVA was done with 5mC antibody (upper row) or without antibody (lower row). Arrows indicate positions of red foci. Scale bar, 5 μm. ( C ) Quantitation of DNA methylation levels of the HIV-1 proviral genome and dynamic range of the HIV EVA signal measurements. Orange: green-to-red EVA signal ratios of HIV locus with (5mC Ab)and without 5mC antibody (no Ab). Blue: HIV EVA was done in the same cells without lambda exonuclease treatment to estimate maximum green signal intensity and technical noise. Different proportions of green oligo were mixed with the same unlabeled oligo (0.00, 0.33, 0.67, 1.00 labeled-to-unlabeled oligo ratios). Each dot represents one cell.

    Techniques Used: Infection, Quantitation Assay, DNA Methylation Assay, Labeling

    18) Product Images from "Functional interactions of DNA topoisomerases with a human replication origin"

    Article Title: Functional interactions of DNA topoisomerases with a human replication origin

    Journal: The EMBO Journal

    doi: 10.1038/sj.emboj.7601578

    Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.
    Figure Legend Snippet: Interaction of topo I and II with the lamin B2 origin in vitro . ( A ) Detection of the in vitro topo I cleavages stabilized on the lower strand by CPT (lane 3), 7-[CH2–Tris] CPT (lane 5) or gimatecan (lane 6), and on the upper strand by CPT (lane 10); lanes 7 and 12: Maxam–Gilbert sequencing reactions; the position of the cleavages also present in vivo is indicated by an asterisk. ( B ) Effect of base substitution mutations in the lamin B2 origin on topo I-mediated cleavage. ( C ) Sequence of the origin portion covered by the replicative complexes; the position of substituted bases is highlighted; the position of in vitro topo I-cleavable complexes is indicated by filled triangles; the asterisks indicate the position of the topo I cleavages also present in vivo . ( D ) Detection of the in vitro VP16-induced topo II cleavages introduced by the enzyme alone (lanes 1–5) or by topo II as part of a complex with nuclear proteins (lanes 6–14); lane 9: the origin DNA incubated with the nuclear extract and VP16 was immunopurified using anti-topo II antibody; black vertical bars indicate the region protected in vivo ; the arrows indicate the borders of the region protected in vitro by the origin binding proteins (OBP) as determined by λ-exonuclease digestion; lanes 10 and 14: Maxam–Gilbert sequencing reactions.

    Techniques Used: In Vitro, Cycling Probe Technology, Sequencing, In Vivo, Incubation, Binding Assay

    19) Product Images from "Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction"

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky154

    The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).
    Figure Legend Snippet: The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).

    Techniques Used: Fluorescence, Standard Deviation, Sequencing, Labeling, Agarose Gel Electrophoresis

    ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.
    Figure Legend Snippet: ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.

    Techniques Used:

    Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.
    Figure Legend Snippet: Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.

    Techniques Used:

    20) Product Images from "Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation"

    Article Title: Upgrading SELEX Technology by Using Lambda Exonuclease Digestion for Single-Stranded DNA Generation

    Journal: Molecules

    doi: 10.3390/molecules15010001

    Digestion of the phosphorylated strand of dsDNA with 25 U lambda exonuclease after PCR with 1 ng template per reaction.
    Figure Legend Snippet: Digestion of the phosphorylated strand of dsDNA with 25 U lambda exonuclease after PCR with 1 ng template per reaction.

    Techniques Used: Polymerase Chain Reaction

    Digestion of the phosphorylated strand of dsDNA with 25 U lambda exonuclease and different digestion times.
    Figure Legend Snippet: Digestion of the phosphorylated strand of dsDNA with 25 U lambda exonuclease and different digestion times.

    Techniques Used:

    21) Product Images from "An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation"

    Article Title: An Optimized Preparation Method for Long ssDNA Donors to Facilitate Quick Knock-In Mouse Generation

    Journal: Cells

    doi: 10.3390/cells10051076

    Schematic diagram of knock-in mouse generation using long ssDNA donors prepared by the phospho-PCR method. ( A ) Flowchart of the phospho-PCR method. A dsDNA template containing a gene cassette of interest (GOI) flanked by homology arms on both sides is depicted at the top. A pair of normal primer and 5′-phospholyrated primer for PCR is used to amplify the starting dsDNA substrates for exonuclease reactions. Strandase Mix A or Lambda exonuclease selectively degrades the phosphorylated chains of the duplex. Strandase Mix B or Exonuclease III subsequently help complete the selective degradations to yield long ssDNAs. Minor PCR products without 5′-phosphorylations are also extensively digested by Exonuclease III activities, whose efficiency is detailed in Results Section 3.2 . Note that this sequential use of two exonucleases enables highly pure ssDNA production. ( B ) Schematic of CRISPR/Cas9 mediated knock-in mouse generation using long ssDNA donors. Long ssDNA donor and Cas9-guide RNA complex can be introduced into mouse fertilized eggs either by pronuclear injection or by electroporation. Treated eggs are transferred into the oviduct of pseudo-pregnant recipients to obtain knock-in founders.
    Figure Legend Snippet: Schematic diagram of knock-in mouse generation using long ssDNA donors prepared by the phospho-PCR method. ( A ) Flowchart of the phospho-PCR method. A dsDNA template containing a gene cassette of interest (GOI) flanked by homology arms on both sides is depicted at the top. A pair of normal primer and 5′-phospholyrated primer for PCR is used to amplify the starting dsDNA substrates for exonuclease reactions. Strandase Mix A or Lambda exonuclease selectively degrades the phosphorylated chains of the duplex. Strandase Mix B or Exonuclease III subsequently help complete the selective degradations to yield long ssDNAs. Minor PCR products without 5′-phosphorylations are also extensively digested by Exonuclease III activities, whose efficiency is detailed in Results Section 3.2 . Note that this sequential use of two exonucleases enables highly pure ssDNA production. ( B ) Schematic of CRISPR/Cas9 mediated knock-in mouse generation using long ssDNA donors. Long ssDNA donor and Cas9-guide RNA complex can be introduced into mouse fertilized eggs either by pronuclear injection or by electroporation. Treated eggs are transferred into the oviduct of pseudo-pregnant recipients to obtain knock-in founders.

    Techniques Used: Knock-In, Polymerase Chain Reaction, CRISPR, Injection, Electroporation

    Purely amplified PCR products and the following sequential use of two exonucleases enabled high-quality ssDNA production. ( A ) Optimization of PCR conditions to obtain a clear single product was the first key step for phospho-PCR-mediated ssDNA production. Primers’ concentrations, annealing temperature, and templates’ concentration for the PCR reaction depicted in Figure 2 B were optimized. The manufacturer’s protocols are underlined, and our optimized conditions are colored in blue. To obtain a single product with high efficiency, the primers’ concentrations should be lower, the annealing temperature should be higher, and the templates’ concentration should be much lower than the manufacturer’s instructions. Asterisks (*) indicate the unwanted non-specific amplifications. Blue triangle indicates the purely amplified products. ( B ) Sequential use of two exonucleases depicted in Figure 2 B allowed for highly pure long ssDNA production. While the single use of Strandase Mix A or Lambda exonuclease selectively degraded 5′-phosphorylated strands to yield ssDNAs (red triangles), dsDNA substrates indicated by asterisks remained in the reaction mixtures. Subsequently added Strandase Mix B or Exonuclease III degraded the remaining dsDNAs to yield pure long ssDNAs. Note that the long ssDNA treated with Exonuclease III was slightly shorter than that treated with Strandase Mix B.
    Figure Legend Snippet: Purely amplified PCR products and the following sequential use of two exonucleases enabled high-quality ssDNA production. ( A ) Optimization of PCR conditions to obtain a clear single product was the first key step for phospho-PCR-mediated ssDNA production. Primers’ concentrations, annealing temperature, and templates’ concentration for the PCR reaction depicted in Figure 2 B were optimized. The manufacturer’s protocols are underlined, and our optimized conditions are colored in blue. To obtain a single product with high efficiency, the primers’ concentrations should be lower, the annealing temperature should be higher, and the templates’ concentration should be much lower than the manufacturer’s instructions. Asterisks (*) indicate the unwanted non-specific amplifications. Blue triangle indicates the purely amplified products. ( B ) Sequential use of two exonucleases depicted in Figure 2 B allowed for highly pure long ssDNA production. While the single use of Strandase Mix A or Lambda exonuclease selectively degraded 5′-phosphorylated strands to yield ssDNAs (red triangles), dsDNA substrates indicated by asterisks remained in the reaction mixtures. Subsequently added Strandase Mix B or Exonuclease III degraded the remaining dsDNAs to yield pure long ssDNAs. Note that the long ssDNA treated with Exonuclease III was slightly shorter than that treated with Strandase Mix B.

    Techniques Used: Amplification, Polymerase Chain Reaction, Concentration Assay

    Mouse Dcx locus as a model site for targeted recombinase knock-in. ( A ) Targeting strategy to insert an iCre recombinase cassette just upstream from the translational stop codon in the Dcx gene is outlined. Schematic of the Dcx gene structure, the guide RNA sequences, and the long ssDNA donor is shown in the upper half. The resulting knock-in allele and its genotyping primers are depicted in the lower half. T2A peptide sequences were employed for bicistronic iCre expressions in Dcx-expressing cells. ( B ) The flowchart of long ssDNA production for Dcx-T2A-iCre knock-in via the phospho-PCR method is depicted. The artificially synthesized gene containing a T2A-iCre cassette was used as the template for phospho-PCR. 5′-phosphorylated strands of the PCR products were sequentially digested by Lambda exonuclease and Exonuclease III to yield long ssDNA donor. ( C ) PCR screening results for knock-in newborns derived from the pronuclear injection shown in Table 2 (Dcx-T2A-iCre) are summarized. Two pairs of primers depicted in ( A ) were used to confirm the designed knock-in. Dcx_Fw and Dcx_Rv were designed outside from the donor DNA’s homology arms to exclude the detection of unintended random integrations. Asterisks indicate the knock-in founders with correct insertions (No.3, No.5, No.6, and No.8). No.5 carried an additional incorrect knock-in allele. ( D ) Boundary sequences between the Dcx gene and T2A-iCre cassette analyzed by using the genome DNA from No.3 founder in ( C ) are aligned.
    Figure Legend Snippet: Mouse Dcx locus as a model site for targeted recombinase knock-in. ( A ) Targeting strategy to insert an iCre recombinase cassette just upstream from the translational stop codon in the Dcx gene is outlined. Schematic of the Dcx gene structure, the guide RNA sequences, and the long ssDNA donor is shown in the upper half. The resulting knock-in allele and its genotyping primers are depicted in the lower half. T2A peptide sequences were employed for bicistronic iCre expressions in Dcx-expressing cells. ( B ) The flowchart of long ssDNA production for Dcx-T2A-iCre knock-in via the phospho-PCR method is depicted. The artificially synthesized gene containing a T2A-iCre cassette was used as the template for phospho-PCR. 5′-phosphorylated strands of the PCR products were sequentially digested by Lambda exonuclease and Exonuclease III to yield long ssDNA donor. ( C ) PCR screening results for knock-in newborns derived from the pronuclear injection shown in Table 2 (Dcx-T2A-iCre) are summarized. Two pairs of primers depicted in ( A ) were used to confirm the designed knock-in. Dcx_Fw and Dcx_Rv were designed outside from the donor DNA’s homology arms to exclude the detection of unintended random integrations. Asterisks indicate the knock-in founders with correct insertions (No.3, No.5, No.6, and No.8). No.5 carried an additional incorrect knock-in allele. ( D ) Boundary sequences between the Dcx gene and T2A-iCre cassette analyzed by using the genome DNA from No.3 founder in ( C ) are aligned.

    Techniques Used: Knock-In, Expressing, Polymerase Chain Reaction, Synthesized, Derivative Assay, Injection

    22) Product Images from "Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template"

    Article Title: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template

    Journal: Synthetic Biology

    doi: 10.1093/synbio/ysaa030

    Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.
    Figure Legend Snippet: Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.

    Techniques Used: Amplification, Introduce, Ligation, Polymerase Chain Reaction

    23) Product Images from "Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis"

    Article Title: Detection of short repeated genomic sequences on metaphase chromosomes using padlock probes and target primed rolling circle DNA synthesis

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-8-103

    In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.
    Figure Legend Snippet: In situ detection of DNA using padlock probes and target primed rolling circle DNA synthesis . (A) The samples are cleaved with a restriction enzyme having a restriction site positioned 3' to the probe binding sequence. It is important that the enzyme does not have any other cleavage sites in close proximity to the 5'-end of the probe binding sequence to avoid degradation of the recognition sequence during exonuclease treatment. (B) The target sequence is made single stranded using the lambda exonuclease which digests duplex DNA in the 5'→3' direction in a highly processive manner, thereby making the target sequence single stranded. (C) The padlock probe is hybridized and ligated on the target sequence. Only padlock probes which are correctly hybridized at the point of ligation will be circularized. (D-E) The rolling circle reaction is initiated by using the target sequence as a primer, thereby locking the rolling circle product to the target sequence. (F) The rolling circle product is visualized by hybridizing a labeled oligonucleotide to the part of the padlock probe not recognizing the genomic hybridization target.

    Techniques Used: In Situ, DNA Synthesis, Binding Assay, Sequencing, Ligation, Labeling, Hybridization

    24) Product Images from "Removal of Spo11 from meiotic DNA breaks in vitro but not in vivo by Tyrosyl DNA Phosphodiesterase 2"

    Article Title: Removal of Spo11 from meiotic DNA breaks in vitro but not in vivo by Tyrosyl DNA Phosphodiesterase 2

    Journal: bioRxiv

    doi: 10.1101/527333

    Ectopic expression of TDP2 cannot remove Spo11 from DSB ends in vivo , but can suppress camptothecin and etoposide sensitivity. a-b, Genomic DNA was isolated at the indicated timepoints from a synchronous meiotic culture of sae2 Δ cells harbouring either an empty vector, or one expressing TDP2 from the ADH1 promoter ( P ADH1 TDP2 ). DNA was purified and digested with Pst I, separated by electrophoresis on a 0.7% agarose gel, blotted to nylon membrane and hybridised with a probe ( MXR2 locus) close to the HIS4 ∷ LEU2 meiotic recombination hotspot. The location of the two major Spo11-DSBs at this locus are marked with solid arrowheads. In sae2 Δ cells, Spo11 is not removed and resection cannot occur, causing DSB species to migrate as a tight band. Expression of TDP2 does not alter this migration pattern (a). b, DNA from the 10 h timepoints was incubated with lambda exonuclease for the indicated length of time. c, The indicated strains harbouring either an empty vector, or one expressing TDP2 from the ADH1 promoter ( P ADH1 TDP2 ) were grown to log phase, serial diluted (10-fold), and spotted onto plates containing 300 µg/ml hygromycin (HYG), to maintain plasmid selection, and the indicated concentrations of etoposide, camptothecin (CPT) or methyl methanesulphonate (MMS), and incubated for 3 days at 30°C.
    Figure Legend Snippet: Ectopic expression of TDP2 cannot remove Spo11 from DSB ends in vivo , but can suppress camptothecin and etoposide sensitivity. a-b, Genomic DNA was isolated at the indicated timepoints from a synchronous meiotic culture of sae2 Δ cells harbouring either an empty vector, or one expressing TDP2 from the ADH1 promoter ( P ADH1 TDP2 ). DNA was purified and digested with Pst I, separated by electrophoresis on a 0.7% agarose gel, blotted to nylon membrane and hybridised with a probe ( MXR2 locus) close to the HIS4 ∷ LEU2 meiotic recombination hotspot. The location of the two major Spo11-DSBs at this locus are marked with solid arrowheads. In sae2 Δ cells, Spo11 is not removed and resection cannot occur, causing DSB species to migrate as a tight band. Expression of TDP2 does not alter this migration pattern (a). b, DNA from the 10 h timepoints was incubated with lambda exonuclease for the indicated length of time. c, The indicated strains harbouring either an empty vector, or one expressing TDP2 from the ADH1 promoter ( P ADH1 TDP2 ) were grown to log phase, serial diluted (10-fold), and spotted onto plates containing 300 µg/ml hygromycin (HYG), to maintain plasmid selection, and the indicated concentrations of etoposide, camptothecin (CPT) or methyl methanesulphonate (MMS), and incubated for 3 days at 30°C.

    Techniques Used: Expressing, In Vivo, Isolation, Plasmid Preparation, Purification, Electrophoresis, Agarose Gel Electrophoresis, Migration, Incubation, Selection

    TDP2 can remove Spo11 peptides from the dsDNA end of natural Spo11-DSBs enabling 5′-3′ resection by lambda exonuclease. a, Schematic of experiment. b, Genomic DNA isolated from meiotic sae2 Δ cells was treated with proteinase K and incubated with lambda exonuclease (λexo) without or with prior treatment with TDP2. DNA was purified and digested with Pst I, separated by electrophoresis on a 0.7% agarose gel, blotted to nylon membrane and hybridised with a probe ( MXR2 locus) close to the well-characterised meiotic recombination hotspot HIS4 ∷ LEU2 . The location of the two major Spo11-DSBs at this locus are marked with solid arrowheads. Disappearance of these signals following TDP2 and lambda exonuclease treatment is due to rapid exonucleolytic degradation from the uncapped Spo11-DSB ends.
    Figure Legend Snippet: TDP2 can remove Spo11 peptides from the dsDNA end of natural Spo11-DSBs enabling 5′-3′ resection by lambda exonuclease. a, Schematic of experiment. b, Genomic DNA isolated from meiotic sae2 Δ cells was treated with proteinase K and incubated with lambda exonuclease (λexo) without or with prior treatment with TDP2. DNA was purified and digested with Pst I, separated by electrophoresis on a 0.7% agarose gel, blotted to nylon membrane and hybridised with a probe ( MXR2 locus) close to the well-characterised meiotic recombination hotspot HIS4 ∷ LEU2 . The location of the two major Spo11-DSBs at this locus are marked with solid arrowheads. Disappearance of these signals following TDP2 and lambda exonuclease treatment is due to rapid exonucleolytic degradation from the uncapped Spo11-DSB ends.

    Techniques Used: Isolation, Incubation, Purification, Electrophoresis, Agarose Gel Electrophoresis

    25) Product Images from "Rapid single-molecule characterisation of nucleic-acid enzymes"

    Article Title: Rapid single-molecule characterisation of nucleic-acid enzymes

    Journal: bioRxiv

    doi: 10.1101/2022.03.03.482895

    λ exonuclease (a) Single-molecule trajectories of DNA degradation by λ exonuclease. (b) Still frames from a recorded movie with the white rectangles numbered 1-3 as a visual guide and marking identical molecules over time, showing their decrease in DNA-stain intensity. Scale bar: 5 μm. (c) Rate distributions of DNA degradation by λ exonuclease at 25°C(yellow, n = 1957 molecules) and 35 °C (grey, n = 648 molecules).
    Figure Legend Snippet: λ exonuclease (a) Single-molecule trajectories of DNA degradation by λ exonuclease. (b) Still frames from a recorded movie with the white rectangles numbered 1-3 as a visual guide and marking identical molecules over time, showing their decrease in DNA-stain intensity. Scale bar: 5 μm. (c) Rate distributions of DNA degradation by λ exonuclease at 25°C(yellow, n = 1957 molecules) and 35 °C (grey, n = 648 molecules).

    Techniques Used: Staining

    26) Product Images from "A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition"

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition

    Journal: Sensors (Basel, Switzerland)

    doi: 10.3390/s18082441

    Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.
    Figure Legend Snippet: Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.

    Techniques Used: Fluorescence

    27) Product Images from "A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells"

    Article Title: A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1205

    ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.
    Figure Legend Snippet: ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Techniques Used: Fluorescence, Activity Assay

    28) Product Images from "Simplified ChIP-exo assays"

    Article Title: Simplified ChIP-exo assays

    Journal: Nature Communications

    doi: 10.1038/s41467-018-05265-7

    ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )
    Figure Legend Snippet: ChIP-exo 5.0 increases library yield. a Schematic of ChIP-exo 5.0. The purple triangle indicates the location of the Read_1 start site, which is also the λ exonuclease stop site. b 2% agarose gel of the electrophoresed library following 18 cycles of PCR for various S. cerevisiae transcription factors assayed by ChIP-exo 1.1 or 5.0. Following ChIP, the sample was split and libraries prepared using the indicated protocols. After splitting the sample, each reaction contained a 50 ml cell equivalent (OD 600 = 0.8) of yeast chromatin, which is five-fold less than the amount optimized for ChIP-exo 1.1. ChIP-exo 5.0 produced greater library yield for all samples. c Heatmaps comparing ChIP-exo 1.1 and 5.0 at the 975 Reb1 primary motifs in a 200 bp window. d Composite plot of data from panel ( c )

    Techniques Used: Chromatin Immunoprecipitation, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Produced

    29) Product Images from "Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases"

    Article Title: Improving Lambda Red Genome Engineering in Escherichia coli via Rational Removal of Endogenous Nucleases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0044638

    Investigating Nuclease Processing with Variably Phosphorothioated Cassettes. A) Diagram of the variably phosphorothioated (VPT) cassette series. Homology arms to lacZ are shown in blue, and the inserted heterologous kanR gene is shown in gold; the lagging-targeting strand is represented as the top strand, and the leading-targeting strand is represented as the bottom strand. The red asterisk indicates the presence of 4 consecutive phosphorotiohate bonds. B) In vitro Lambda Exo digestion of non-phosphorothioated (VPT1, left), single end phosphorothioated (VPT2, center), and dually phosphorothioated (VPT4, right) dsDNA cassettes. Samples were digested with no enzyme (lanes 1, 5, 9) and tenfold-increasing amounts of enzyme, from left to right. The bottom band is dsDNA, and the top band is the ssDNA product after Lambda Exo degrades one of the two strands. PAGE analysis confirms that dually phosphorothioated cassettes are protected from degradation. C–E) Recombination frequencies for the VPT cassette series in EcNR2 (C), nuc4 − (D), and EcNR2. xseA − (E). Insertion frequencies were measured as the number of kanamycin resistant recombinants over the total number of cfu (as plated on non-selective media). These data are presented as the mean with the error bars as standard deviation; n = 3 for EcNR2 and EcNR2. xseA − , and n = 2 for nuc4 − . Numerical data are shown in Table S2 . F G) Cassette insertion frequencies were measured in technical replicates for EcNR2, nuc4 − , and EcNR2. xseA − . Data in F) G) are presented as the mean with the standard error of the mean. The ratio between VPT4 and VPT1 (F) indicates a strain’s ability to recombine cassettes with terminal PT bonds preventing the direct action of Lambda Exo. The ratio between VPT4 and VPT7 (G) indicates whether a strain is more able to process terminal PT bonds (VPT4) in comparison with internal PT bonds (VPT7).
    Figure Legend Snippet: Investigating Nuclease Processing with Variably Phosphorothioated Cassettes. A) Diagram of the variably phosphorothioated (VPT) cassette series. Homology arms to lacZ are shown in blue, and the inserted heterologous kanR gene is shown in gold; the lagging-targeting strand is represented as the top strand, and the leading-targeting strand is represented as the bottom strand. The red asterisk indicates the presence of 4 consecutive phosphorotiohate bonds. B) In vitro Lambda Exo digestion of non-phosphorothioated (VPT1, left), single end phosphorothioated (VPT2, center), and dually phosphorothioated (VPT4, right) dsDNA cassettes. Samples were digested with no enzyme (lanes 1, 5, 9) and tenfold-increasing amounts of enzyme, from left to right. The bottom band is dsDNA, and the top band is the ssDNA product after Lambda Exo degrades one of the two strands. PAGE analysis confirms that dually phosphorothioated cassettes are protected from degradation. C–E) Recombination frequencies for the VPT cassette series in EcNR2 (C), nuc4 − (D), and EcNR2. xseA − (E). Insertion frequencies were measured as the number of kanamycin resistant recombinants over the total number of cfu (as plated on non-selective media). These data are presented as the mean with the error bars as standard deviation; n = 3 for EcNR2 and EcNR2. xseA − , and n = 2 for nuc4 − . Numerical data are shown in Table S2 . F G) Cassette insertion frequencies were measured in technical replicates for EcNR2, nuc4 − , and EcNR2. xseA − . Data in F) G) are presented as the mean with the standard error of the mean. The ratio between VPT4 and VPT1 (F) indicates a strain’s ability to recombine cassettes with terminal PT bonds preventing the direct action of Lambda Exo. The ratio between VPT4 and VPT7 (G) indicates whether a strain is more able to process terminal PT bonds (VPT4) in comparison with internal PT bonds (VPT7).

    Techniques Used: In Vitro, Polyacrylamide Gel Electrophoresis, Standard Deviation

    30) Product Images from "DSBCapture: in situ capture and direct sequencing of dsDNA breaks"

    Article Title: DSBCapture: in situ capture and direct sequencing of dsDNA breaks

    Journal: Nature methods

    doi: 10.1038/nmeth.3960

    DSBCapture methodology and validation ( a ) DSBCapture workflow. (1) DSBs in fixed nuclei were blunt-end repaired, (2) A-tailed and (3) ligated to a biotinylated (black ball) modified P5 Illumina adapter (orange lines). (4) Excess adapters were removed by lambda exonuclease digestion; (5) DNA extracted from lysed nuclei was fragmented by sonication, (6) bead-captured (hollow ball) and blunted-end repaired, (7) A-tailed, and (8) ligated to a modified P7 Illumina adapter (purple lines). (9) Captured break sites were PCR amplified, (10) size selected, (11) quantified and (12) sequenced. Sequences of the DSBCapture adapters: modified P5 Illumina adapter and modified P7 Illumina adapter (B = biotin; P = phosphorylated; * = phosphorothioate bond). ( b ) DSBs created by EcoRV cleavage in fixed nuclei (N = 1). PCR duplicates have been removed. Data range is shown in square brackets and black boxes illustrate the genomic location of EcoRV sites. A 20 kb region and a 110 bp region are shown. Pink and purple lines: reads from the sense and antisense strand, respectively. As EcoRV is a blunt cutter, reads originate directly from the cleavage site. ( c ) AsiSI cleavage sites (black boxes) detected by DSBCapture (N = 1). Cleavage by AsiSI generates a 2 bp 3’ overhang; end processing removes this overhang generating the 2 bp gap in the center of the peak. A 2 kb and a 200 bp region are shown. ( d ) Venn diagram illustrating the overlap of DSBs detected at AsiSI sites by DSBCapture and γH2AX ChIP-seq 9 .
    Figure Legend Snippet: DSBCapture methodology and validation ( a ) DSBCapture workflow. (1) DSBs in fixed nuclei were blunt-end repaired, (2) A-tailed and (3) ligated to a biotinylated (black ball) modified P5 Illumina adapter (orange lines). (4) Excess adapters were removed by lambda exonuclease digestion; (5) DNA extracted from lysed nuclei was fragmented by sonication, (6) bead-captured (hollow ball) and blunted-end repaired, (7) A-tailed, and (8) ligated to a modified P7 Illumina adapter (purple lines). (9) Captured break sites were PCR amplified, (10) size selected, (11) quantified and (12) sequenced. Sequences of the DSBCapture adapters: modified P5 Illumina adapter and modified P7 Illumina adapter (B = biotin; P = phosphorylated; * = phosphorothioate bond). ( b ) DSBs created by EcoRV cleavage in fixed nuclei (N = 1). PCR duplicates have been removed. Data range is shown in square brackets and black boxes illustrate the genomic location of EcoRV sites. A 20 kb region and a 110 bp region are shown. Pink and purple lines: reads from the sense and antisense strand, respectively. As EcoRV is a blunt cutter, reads originate directly from the cleavage site. ( c ) AsiSI cleavage sites (black boxes) detected by DSBCapture (N = 1). Cleavage by AsiSI generates a 2 bp 3’ overhang; end processing removes this overhang generating the 2 bp gap in the center of the peak. A 2 kb and a 200 bp region are shown. ( d ) Venn diagram illustrating the overlap of DSBs detected at AsiSI sites by DSBCapture and γH2AX ChIP-seq 9 .

    Techniques Used: Modification, Sonication, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation

    31) Product Images from "MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling"

    Article Title: MassCode Liquid Arrays as a Tool for Multiplexed High-Throughput Genetic Profiling

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0018967

    Lambda exonuclease mediates efficient MCT hybrid synthesis. A) Bioanalyzer DNA 1000 capillary electrophoresis analysis of a MC-PCR sample subjected to lambda exonuclease and/or 418 amu MCT probes during hybrid synthesis reactions. Alien DNA served as template for MC-PCR resulting in amplicons (209 bp) encoded on one end by a 352 amu MCT derived from forward priming and modified on the other end by phosphate derived from reverse priming. Aliquots of pooled amplicons served as sample input for hybrid synthesis reactions for lanes 1–4 under the conditions specified. Aliquots of a pooled MC-PCR without Alien DNA served as no template controls (NTC) for each hybrid synthesis reaction condition. The molarity in nanomolar of the Alien amplicons present at the end of the hybrid synthesis process is shown under each band. Alien hybrids possess a 103 bp double stranded segment and a single stranded segment of 106 bases. They cannot be accurately sized or quantified by CE. B) MCT detection from the molecular species formed during the hybrid synthesis reactions described in panel A. Results from NTC samples (a) were compared to those of samples containing DNA (b) for each condition tested. Gray bars, measuring forward primer binding/extension, show the response levels detected by the mass analyzer at 353 amu [M+H] + ; red bars, measuring probe binding/extension, show the 419 amu [M+H] + response levels. L, DNA ladder.
    Figure Legend Snippet: Lambda exonuclease mediates efficient MCT hybrid synthesis. A) Bioanalyzer DNA 1000 capillary electrophoresis analysis of a MC-PCR sample subjected to lambda exonuclease and/or 418 amu MCT probes during hybrid synthesis reactions. Alien DNA served as template for MC-PCR resulting in amplicons (209 bp) encoded on one end by a 352 amu MCT derived from forward priming and modified on the other end by phosphate derived from reverse priming. Aliquots of pooled amplicons served as sample input for hybrid synthesis reactions for lanes 1–4 under the conditions specified. Aliquots of a pooled MC-PCR without Alien DNA served as no template controls (NTC) for each hybrid synthesis reaction condition. The molarity in nanomolar of the Alien amplicons present at the end of the hybrid synthesis process is shown under each band. Alien hybrids possess a 103 bp double stranded segment and a single stranded segment of 106 bases. They cannot be accurately sized or quantified by CE. B) MCT detection from the molecular species formed during the hybrid synthesis reactions described in panel A. Results from NTC samples (a) were compared to those of samples containing DNA (b) for each condition tested. Gray bars, measuring forward primer binding/extension, show the response levels detected by the mass analyzer at 353 amu [M+H] + ; red bars, measuring probe binding/extension, show the 419 amu [M+H] + response levels. L, DNA ladder.

    Techniques Used: Electrophoresis, Polymerase Chain Reaction, Derivative Assay, Modification, Binding Assay

    Overview of MassCode probe array assay format. A) The MassCode workflow begins with a MC-PCR reaction containing all primer pairs for each target group. One primer is labeled with MCT, the other with phosphate. Anti-sense primers are phosphate labeled if anti-sense probes are used in the ensuing step, and vice versa. B) MC-PCR products containing 5′ phosphate strands experience specific digestion of those strands at 37°C after the addition of an admixture containing lambda exonuclease. Single-stranded amplified target DNA labeled with one MCT remains, but duplex off-target DNA labeled with two MCTs and single-stranded off-target DNA labeled with one MCT also remain due to mispriming events during the multiplex PCR (not shown). Directly after digestion a second round of selection is performed during one PCR-like cycle. MassCode probes are annealed to internal sequence of the single-stranded target amplicons and serve as extension primers for DNA polymerase, the result is a double-strand single-strand segmented hybrid labeled with two unique MassCode reporters. Digestion and probing chemistry are combined into a single reagent that is added to the MC-PCR tube, making the process amenable to automation. C) Unincorporated oligonucleotides and misprimed amplified DNA less than 100 bp are removed during a reaction clean-up step. D) and E) MCTs are cleaved from the hybrids upon exposure to UV light and flowed directly into a single quadrupole mass spectrometer for detection.
    Figure Legend Snippet: Overview of MassCode probe array assay format. A) The MassCode workflow begins with a MC-PCR reaction containing all primer pairs for each target group. One primer is labeled with MCT, the other with phosphate. Anti-sense primers are phosphate labeled if anti-sense probes are used in the ensuing step, and vice versa. B) MC-PCR products containing 5′ phosphate strands experience specific digestion of those strands at 37°C after the addition of an admixture containing lambda exonuclease. Single-stranded amplified target DNA labeled with one MCT remains, but duplex off-target DNA labeled with two MCTs and single-stranded off-target DNA labeled with one MCT also remain due to mispriming events during the multiplex PCR (not shown). Directly after digestion a second round of selection is performed during one PCR-like cycle. MassCode probes are annealed to internal sequence of the single-stranded target amplicons and serve as extension primers for DNA polymerase, the result is a double-strand single-strand segmented hybrid labeled with two unique MassCode reporters. Digestion and probing chemistry are combined into a single reagent that is added to the MC-PCR tube, making the process amenable to automation. C) Unincorporated oligonucleotides and misprimed amplified DNA less than 100 bp are removed during a reaction clean-up step. D) and E) MCTs are cleaved from the hybrids upon exposure to UV light and flowed directly into a single quadrupole mass spectrometer for detection.

    Techniques Used: Polymerase Chain Reaction, Labeling, Amplification, Multiplex Assay, Selection, Sequencing, Mass Spectrometry

    32) Product Images from "Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template"

    Article Title: Synthesis of libraries and multi-site mutagenesis using a PCR-derived, dU-containing template

    Journal: Synthetic Biology

    doi: 10.1093/synbio/ysaa030

    Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.
    Figure Legend Snippet: Schematic overview of the SLUPT strategy. Step 1: The gene of interest is amplified with a 5′ phosphorylated top strand primer and dNTP’s containing dU (blue). The primer for the bottom strand is not phosphorylated. Optional, nonhomologous regions (e.g. to introduce restriction enzyme sites) are shown in green. Step 2: The phosphorylated strand is selectively degraded by lambda exonuclease to create the uracil-containing single stranded template. Step 3: An end-primer complementary to the 3′ terminus and 5′ phosphorylated internal primers containing altered bases are annealed to the uracil containing single strand template. Altered bases depicted as X’s in red box. Gap filling and ligation are performed by Phusion-U and Taq ligase to create a mutated, complementary strand. Step 4: The Uracil-containing single stranded template is digested by UDG. Step 5: The single-stranded product is made double stranded and amplified by PCR.

    Techniques Used: Amplification, Introduce, Ligation, Polymerase Chain Reaction

    33) Product Images from "ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints"

    Article Title: ChIP-nexus: a novel ChIP-exo protocol for improved detection of in vivo transcription factor binding footprints

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3121

    Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.
    Figure Legend Snippet: Superior performance of ChIP-nexus in discovering relevant binding footprints for transcription factors (a) Outline of ChIP-nexus 1) The transcription factor of interest (brown) is immunoprecipitated from chromatin fragments with antibodies in the same way as during conventional ChIP-seq experiments. 2) While still bound to the antibodies, the DNA ends are repaired, dA-tailed and then ligated to a special adaptor that contains a pair of sequences for library amplification (arrows indicate the correct orientation for them to be functional), a BamHI site (black dot) for linearization, and a 9-nucleotide barcode containing 5 random bases and 4 fixed bases to remove reads resulting from over-amplification of library DNA. The barcode is part of a 5′ overhang, which reduces adaptor-adaptor ligation. 3) After the adaptor ligation step, the 5′ overhang is filled, copying the random barcode and generating blunt ends for lambda exonuclease digestion. 4) Lambda exonuclease (blue Pacman) digests until it encounters a physical barrier such as a cross-linked protein-DNA complex (‘Do not enter’ sign = ‘stop base’). 5) Single-stranded DNA is eluted and purified. 6) Self-circularization places the barcode next to the ‘stop base’. 7) An oligonucleotide (red arc) is paired with the region around the BamHI site for BamHI digestion (black scissors). 8) The digestion results in re-linearized DNA fragments with suitable Illumina sequences on both ends, ready for PCR library amplification. 9) Using single-end sequencing with the standard Illumina primer, each fragment is sequenced: first the barcode, then the genomic sequence starting with the ‘stop base’. 10) After alignment of the genomic sequences, reads with identical start positions and identical barcodes are removed. The final output is the position, number and strand orientation of the ‘stop’ bases. The frequencies of ‘stop’ bases on the positive strand are shown in red, while those on the negative strand are shown in blue. (b–e) Comparison of conventional ChIP-seq data (extended reads), ChIP-nexus data (raw stop base reads) and data generated using the original ChIP-exo protocol (raw stop base reads). (b) TBP profiles in human K562 cells at the RPS12 promoter. Although ChIP-nexus and ChIP-exo generally agree on TBP binding footprints, ChIP-nexus provides better coverage and richer details than ChIP-exo, which shows signs of over-amplification as large numbers of reads accumulate at a few discreet bases. (c) Dorsal profiles at the D. melanogaster decapentaplegic (dpp) enhancer. Five “Strong” dorsal binding sites (S1–S5) were previously mapped by in vitro DNase footprinting 12 . Note that ChIP-nexus identifies S4 as the only site with significant Dorsal binding in vivo . At the same time, ChIP-exo performed by Peconic did not detect any clear Dorsal footprint within the enhancer, in part due to the low read counts obtained. (d) Dorsal profiles at the rhomboid (rho) NEE enhancer. Four Dorsal binding sites (d1–d4) were previously mapped by in vitro DNase footprinting 14 . Note that ChIP-nexus identifies d3 as the strongest dorsal binding site in vivo , consistent with its close proximity to two Twist binding sites. Again, the original ChIP-exo protocol did not detect any clear Dorsal footprint within the enhancer. (e) Twist profiles at the same rho enhancer. Note that ChIP-nexus shows strong Twist footprints surrounding the two Twist binding sites (t1, t2) 14 . In this case, ChIP-exo performed by Peconic identified a similar Twist footprint. This shows that the Peconic experiments, which were performed with the same chromatin extracts as the Dorsal experiments, worked in principle but were less robust than our ChIP-nexus experiments.

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Amplification, Functional Assay, Ligation, Purification, Polymerase Chain Reaction, Sequencing, Genomic Sequencing, Generated, In Vitro, Footprinting, In Vivo

    Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.
    Figure Legend Snippet: Analysis of the Dorsal, Twist and Max in vivo footprint (a–c) For each factor, the top 200 motifs with the highest ChIP-nexus read counts were selected and are shown in descending order as heat map. The footprints show a consistent boundary on the positive strand (red) and negative strand (blue) around each motif. The zoomed-in average profile below reveals that the footprints are wider than the motif. A schematic representation of the digestion pattern is shown below using Pacman symbols for lambda exonuclease. (a) The ChIP-nexus footprint for Dorsal (NFkB) on its canonical motif (GGRWWTTCC with up to one mismatch) extends on average 5 bp away from the motif edge. Thus, the average dorsal footprint is 18 bp long (horizontal black bar). (b) The Twist ChIP-nexus footprint on the E-box motif CABATG (no mismatch) has two outside boundaries, one at 11 bp, and one at 2 bp away from the motif edge, suggesting interactions with flanking DNA sequences. Each portion of the footprint is around 8–9bp long (horizontal black bar). (c) The Max ChIP-nexus footprint on its canonical E-box motif (CACGTG, no mismatch) has an outside boundary at 8 bp away from the motif edge, as well as a boundary inside the motif (at the A/T base), suggesting two partial footprints (horizontal black bars). (d, e) Average Max and Twist ChIP-nexus footprints at the top 200 sites for all possible E-box variants (CANNTG). Each variant profile includes its reverse complement. (d) Max binds specifically to the canonical CACGTG motif and to a lesser extent to the CACATG motif. Note that the Max footprint shape looks identical between the two motifs. (e) In contrast, the Twist binding specificity and the footprint shape is more complex. Notably, the outer boundary at -11bp is stronger at the CATATG and CACATG motif, whereas the inner boundary at -2 bp is stronger at the CAGATG motif.

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Variant Assay, Binding Assay

    34) Product Images from "Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH"

    Article Title: Triplex-forming properties and enzymatic incorporation of a base-modified nucleotide capable of duplex DNA recognition at neutral pH

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab572

    Template-directed assembly of Z-TFOs. ( A ) Strategy for the assembly of oligonucleotides containing a triplex-forming sequence (TFS; shown in red) containing Z. (i) Primer extension of a template that contains the W–C complement to the TFO (underlined) allows the incorporation of Z by the formation of a GZ base pair at high pH (and the absence of dCTP). (ii) The modified oligonucleotide can then be isolated by selective degradation of the phosphate-labelled template (in yellow) by the action of lambda exonuclease. ( B ) Electrophoretic mobility shift assay showing the products of the extension and digestion of such reactions. The composition of each sample is shown above each lane of the gel. Final concentration of the strands and dNTPS was 5 and 100 μM, respectively. Extension reactions were performed for 2 h at 72°C with 2 units of Therminator Pol, whilst digestion reactions were performed for 2 h with 10 units of Lambda exonuclease at 37°C. The complexes were then separated on a 15% non-denaturing polyacrylamide gel and subjected to post-staining with GelRed.
    Figure Legend Snippet: Template-directed assembly of Z-TFOs. ( A ) Strategy for the assembly of oligonucleotides containing a triplex-forming sequence (TFS; shown in red) containing Z. (i) Primer extension of a template that contains the W–C complement to the TFO (underlined) allows the incorporation of Z by the formation of a GZ base pair at high pH (and the absence of dCTP). (ii) The modified oligonucleotide can then be isolated by selective degradation of the phosphate-labelled template (in yellow) by the action of lambda exonuclease. ( B ) Electrophoretic mobility shift assay showing the products of the extension and digestion of such reactions. The composition of each sample is shown above each lane of the gel. Final concentration of the strands and dNTPS was 5 and 100 μM, respectively. Extension reactions were performed for 2 h at 72°C with 2 units of Therminator Pol, whilst digestion reactions were performed for 2 h with 10 units of Lambda exonuclease at 37°C. The complexes were then separated on a 15% non-denaturing polyacrylamide gel and subjected to post-staining with GelRed.

    Techniques Used: Sequencing, Modification, Isolation, Electrophoretic Mobility Shift Assay, Concentration Assay, Staining

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    New England Biolabs λ exonuclease buffer
    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and <t>λ-exonuclease</t> treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.
    λ Exonuclease Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    New England Biolabs λ exo
    The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with <t>λ</t> exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).
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    Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Journal: Journal of Virology

    Article Title: Simultaneous Single-Cell In Situ Analysis of Human Adenovirus Type 5 DNA and mRNA Expression Patterns in Lytic and Persistent Infection

    doi: 10.1128/JVI.00166-17

    Figure Lengend Snippet: Concurrent in situ detection of HAdV-5 DNA and mRNAs. (A) HeLa cells were infected with HAdV-5 (10 FFU/cell) and analyzed at 25 hpi. HAdV-5 genomic DNA is presented in magenta (image A), E1A mRNAs ( 13S and 12S ) in green (image B), MLTU mRNAs (exon I_II and exon II_III) in red (image C), and ACTB mRNA in yellow (image D). A merged image (image E) and uninfected cells (image F) also are shown. (B) Detection of spliced E1A mRNAs (13S and 12S), MLTU mRNAs (exon I_II and exon II_III), and ACTB mRNA with PLPs in HeLa cells 25 hpi according to the standard protocol (images A and C). Reverse transcriptase was omitted from the cDNA synthesis reaction (images B and D). Detection of HAdV-5 DNA with PLP in HeLa cells 25 hpi was performed according to the standard protocol (image E). MscI endonuclease and λ-exonuclease treatments were omitted during HAdV-5 DNA preparation (image F). Scale bar, 50 μm.

    Article Snippet: Finally, dsDNA fragments were converted to single-stranded DNA (ssDNA) in 1× λ-exonuclease buffer (NEB) with 10% glycerol, 0.2 μg/μl BSA, and 0.2 U/μl λ-exonuclease (NEB) for 30 min at 37°C.

    Techniques: In Situ, Infection, Plasmid Purification

    Expansion of HiLands-P upon lamin loss A. A plot of log 2 fold increased or decreased total interactions between two HiLands-P regions upon lamin loss as a function of the distance between the regions. B. FISH probe production. PCR1 amplifies the probes for a specific sub-library using the indicated sub-library primers. PCR2 produces the labeled sub-library probes using the fluorescently labeled and phosphorylated common primers. Lambda exonuclease digests the phosphorylated DNA strand to produce the single stranded DNA probes for FISH. C. Four regions (dashed boxes) on Chromosome 1, 4, 13, and 14 consisting of mostly HiLands-P were selected for FISH. HiLands are shown in corresponding colors. D. Box plot showing the log 2 fold change of inter-TAD interactions for 20-Kb windows in the whole genome (All) or in selected chromosome regions shown in C. Only HiLands-P interactions are included. E. Two representative 3D-projection FISH images for each of the four selected regions in C. Purple: DAPI staining for DNA. White: FISH signal. The white dashed lines demarcate the boundaries of nuclei that are next to one another. Scale bars, 5 µm. The volume and surface areas of the four chromatin regions are quantified to the right. P-values, Wilcoxon rank-sum test.

    Journal: bioRxiv

    Article Title: Lamins organize the global three-dimensional genome from the nuclear periphery

    doi: 10.1101/211656

    Figure Lengend Snippet: Expansion of HiLands-P upon lamin loss A. A plot of log 2 fold increased or decreased total interactions between two HiLands-P regions upon lamin loss as a function of the distance between the regions. B. FISH probe production. PCR1 amplifies the probes for a specific sub-library using the indicated sub-library primers. PCR2 produces the labeled sub-library probes using the fluorescently labeled and phosphorylated common primers. Lambda exonuclease digests the phosphorylated DNA strand to produce the single stranded DNA probes for FISH. C. Four regions (dashed boxes) on Chromosome 1, 4, 13, and 14 consisting of mostly HiLands-P were selected for FISH. HiLands are shown in corresponding colors. D. Box plot showing the log 2 fold change of inter-TAD interactions for 20-Kb windows in the whole genome (All) or in selected chromosome regions shown in C. Only HiLands-P interactions are included. E. Two representative 3D-projection FISH images for each of the four selected regions in C. Purple: DAPI staining for DNA. White: FISH signal. The white dashed lines demarcate the boundaries of nuclei that are next to one another. Scale bars, 5 µm. The volume and surface areas of the four chromatin regions are quantified to the right. P-values, Wilcoxon rank-sum test.

    Article Snippet: The second PCR products were purified using Zymo DNA clean & Concentrator-500 columns (Zymo Research, D4032) and then treated with lambda exonuclease (NEB, M0262L) to remove the unlabeled strand and to produce the single strand DNA probes (ssDNA).

    Techniques: Fluorescence In Situ Hybridization, Labeling, Staining

    Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

    Journal: Nucleic Acids Research

    Article Title: A low-bias and sensitive small RNA library preparation method using randomized splint ligation

    doi: 10.1093/nar/gkaa480

    Figure Lengend Snippet: Schematic of randomized splint ligation library preparation. First the preadenylated 3′ adapter is ligated on using randomized splint ligation. Following adapter ligation, the excess adapter is depleted using 5′ deadenylase and lambda exonuclease, and the degenerate portion of the adapter is cleaved off by excising the deoxyuracil using USER. Next the 5′ adapter is ligated on using randomized splint ligation and cDNA is synthesized using the remaining portion of the 3′ adapter splint strand as a primer for the reverse transcription. Finally, library molecules containing both adapters are enriched and extended using PCR.

    Article Snippet: These reactions were incubated in a thermocycler at 25°C for 1 h. Following ligation 2.5 units of lambda exonuclease (NEB M0262) and 25 units of 5′ deadenylase (NEB M0331) were added and the reactions were incubated for 15 min at 30°C, 15 min at 37°C and 5 min at 75°C.

    Techniques: Ligation, Synthesized, Polymerase Chain Reaction

    The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).

    Journal: Nucleic Acids Research

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    doi: 10.1093/nar/gky154

    Figure Lengend Snippet: The digestion of different 5′-OH DNA duplexes by λ exo. ( A ) The relative digestion rates of P1-5’OH-6FAM duplexes. The rate of fluorescence increase of P1-5’OH-6FAM/C1-PM(42) is set to 1.0. Error bars represent the standard deviation from experiments performed in triplicate. ( B ) The schematic structures of different P1-5’OH-6FAM duplexes. The complete sequence of the P1-5’OH-6FAM/C1-PM(42) duplex is shown. In the schematic structures, only the first seven base pairs from the 5′ end of the labeled probe are shown to highlight the variation of the sequences near 5′ end in comparison with the P1-5’OH-6FAM/C1-PM(42) duplex. Other parts of all the tested duplexes are identical. The mismatched bases are indicated in red in the tested mismatched DNA duplexes. ( C ) Agarose gel electrophoresis results for the duplexes after reacting with λ exo for different period of time. (i) P1-5’OH-6FAM/C1-2-mis(42) (lane 1, 3, 5 and 7), P1-5’OH-6FAM/C1-PM(42) (lanes 2, 4, 6 and 8), (ii) P1’-5’OH-unlabeled/C1-2-mis(42) (lane 1, 3, 5 and 7) and P1’-5’OH-unlabeled/C1-PM(42) (lanes 2, 4, 6 and 8).

    Article Snippet: This was confirmed in our work by the measurement of the digestion rates of P9-5’PO4 -15FAM/C2-PM(42) by normal λ exo (WT-1 from NEB) and the R28A variant that we expressed (Figure ).

    Techniques: Fluorescence, Standard Deviation, Sequencing, Labeling, Agarose Gel Electrophoresis

    The relative rates of fluorescence increase of the P9-5’PO 4 -15FAM/C2-PM(42), P9-5’PO 4 -15FAM/C2-2-mis(42), P5-5’C6-15FAM/C2-PM(42), P5-5’C6-15FAM/C2-2-mis(42), P10-5’FAM/C2-PM(42) and P10-5’FAM/C2-2-mis(42) by λ exo WT-1 and R28A variant at different enzyme concentrations. The rate of fluorescence increase of P9-5’PO 4 -15FAM/C2-PM(42) digested by 3.6 nM of λ exo WT-1 was set to 1.0. The reaction processes of the three duplexes are schematically depicted.

    Journal: Nucleic Acids Research

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    doi: 10.1093/nar/gky154

    Figure Lengend Snippet: The relative rates of fluorescence increase of the P9-5’PO 4 -15FAM/C2-PM(42), P9-5’PO 4 -15FAM/C2-2-mis(42), P5-5’C6-15FAM/C2-PM(42), P5-5’C6-15FAM/C2-2-mis(42), P10-5’FAM/C2-PM(42) and P10-5’FAM/C2-2-mis(42) by λ exo WT-1 and R28A variant at different enzyme concentrations. The rate of fluorescence increase of P9-5’PO 4 -15FAM/C2-PM(42) digested by 3.6 nM of λ exo WT-1 was set to 1.0. The reaction processes of the three duplexes are schematically depicted.

    Article Snippet: This was confirmed in our work by the measurement of the digestion rates of P9-5’PO4 -15FAM/C2-PM(42) by normal λ exo (WT-1 from NEB) and the R28A variant that we expressed (Figure ).

    Techniques: Fluorescence, Variant Assay

    ( A ) Schematic depiction of the principle of the method used to investigate the reactions between λ exo and different dsDNA substrates. ( B ) The proposed hydrophobic interaction area (green) and π–π stacking interaction area (yellow) marked in the crystal structure of λ exo with 5′-PO 4 DNA duplex (PDB 3SM4). The 5′ end of the DNA strand was indicated in red. ( C ) The proposed hydrophobic interaction between the hydrophobic amino acid residues (green) in λ exo and the C6 spacer modification (grey) at the 5′ end of the DNA strand (red). ( D ) The proposed π–π stacking interaction between the amino acid residues (yellow) in λ exo and the FAM tag (gray) labeled at the third nucleotide from the 5′ end of the DNA strand (red). R28 was shown in purple (with the N atoms in blue) in (B), (C) and (D). The dataset of the protein-DNA complex structure was obtained from PDB 3SM4 and drawn with Pymol. The C6 spacer and FAM were drawn with Pymol.

    Journal: Nucleic Acids Research

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    doi: 10.1093/nar/gky154

    Figure Lengend Snippet: ( A ) Schematic depiction of the principle of the method used to investigate the reactions between λ exo and different dsDNA substrates. ( B ) The proposed hydrophobic interaction area (green) and π–π stacking interaction area (yellow) marked in the crystal structure of λ exo with 5′-PO 4 DNA duplex (PDB 3SM4). The 5′ end of the DNA strand was indicated in red. ( C ) The proposed hydrophobic interaction between the hydrophobic amino acid residues (green) in λ exo and the C6 spacer modification (grey) at the 5′ end of the DNA strand (red). ( D ) The proposed π–π stacking interaction between the amino acid residues (yellow) in λ exo and the FAM tag (gray) labeled at the third nucleotide from the 5′ end of the DNA strand (red). R28 was shown in purple (with the N atoms in blue) in (B), (C) and (D). The dataset of the protein-DNA complex structure was obtained from PDB 3SM4 and drawn with Pymol. The C6 spacer and FAM were drawn with Pymol.

    Article Snippet: This was confirmed in our work by the measurement of the digestion rates of P9-5’PO4 -15FAM/C2-PM(42) by normal λ exo (WT-1 from NEB) and the R28A variant that we expressed (Figure ).

    Techniques: Modification, Labeling

    ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.

    Journal: Nucleic Acids Research

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    doi: 10.1093/nar/gky154

    Figure Lengend Snippet: ( A ) Time courses of the digestion reactions of P11-5’OH-dSpacer-15FAM-duplexes by λ exo. The schematic structure of P11-5’OH-dSpacer-15FAM duplexes and 5’-dSpacer are shown. ( B ) Time courses of the digestion reactions of P3-5’OH-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM-duplexes are shown.

    Article Snippet: This was confirmed in our work by the measurement of the digestion rates of P9-5’PO4 -15FAM/C2-PM(42) by normal λ exo (WT-1 from NEB) and the R28A variant that we expressed (Figure ).

    Techniques:

    Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.

    Journal: Nucleic Acids Research

    Article Title: Noncanonical substrate preference of lambda exonuclease for 5′-nonphosphate-ended dsDNA and a mismatch-induced acceleration effect on the enzymatic reaction

    doi: 10.1093/nar/gky154

    Figure Lengend Snippet: Time courses of the digestion reactions of P3-5’OH-15FAM and P5-5’C6-15FAM duplexes by λ exo. The schematic structures of P3-5’OH-15FAM/C2-2-mis(42), P3-5’OH-15FAM/C2-2-mis(42) and 5’C6 spacer are shown. The mismatched bases are indicated in red.

    Article Snippet: This was confirmed in our work by the measurement of the digestion rates of P9-5’PO4 -15FAM/C2-PM(42) by normal λ exo (WT-1 from NEB) and the R28A variant that we expressed (Figure ).

    Techniques: