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    1) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    2) Product Images from "Secondary structure formation and DNA instability at fragile site FRA16B"

    Article Title: Secondary structure formation and DNA instability at fragile site FRA16B

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp1245

    Analysis of FRA16B replication efficiency and instability in human cells using an SV40 replication system. ( A ) Diagram of replication constructs used for replication efficiency and instability assays. The bidirectional SV40 ori (open circles) was cloned into pGEM3zf(+) to generate pGEM-SV40 ori , used as a control. The 921-bp FRA16B fragment (shaded box) was inserted 30, 300, 400 or 700 bp from the SV40 ori , and in one of two replication orientations (represented by arrows). ( B ) Schematic of strategy used to determine the replication efficiency of FRA16B constructs and instability events following replication in HEK293T cells. To determine replication efficiency, equal amounts of pGEM-SV40 ori and each FRA16B-containing construct were co-transfected into HEK293T cells. After replication for 48 h, SV40 DNAs were extracted, purified and digested with DpnI to remove any unreplicated templates. Replicated molecules were then subjected to Southern blot analysis. To examine FRA16B-associated instability, each FRA16B-containing construct was transfected and replicated in HEK293T cells. SV40 DNA was extracted, purified and digested with DpnI to remove any unreplicated templates. These DNAs were transformed into E. coli , isolated, and digested with restriction enzymes to release the FRA16B insert. Replication products were analyzed on 1.5% agarose gels to score insertions and deletions. As a control, replication constructs were directly transformed into E. coli , without replication in human cells, to account for background instability in E. coli . ( C ) Replication efficiency of FRA16B constructs in HEK293T cells. The replication efficiency of each FRA16B-containing construct was determined by the ratio of replicated FRA16B DNA to the control (pGEM-SV40 ori ). Results are shown as the mean ± SD from six individual experiments. ( D ) Identification of mutation events of replication in HEK293T cells using gel electrophoresis. A representative agarose gel shows individual HEK293T cell replication products from clone 17. Replication products (#4 and #8) exhibit large base pair deletions, as demonstrated by faster migrating products (asterisks) compared to unreplicated DNA (designated as C). M, molecular weight marker. ( E ) Mutation analysis of FRA16B constructs with different replication orientations and distances from the SV40 ori . After replication in HEK293T cells, purified SV40 DNA was digested to eliminate any unreplicated parental templates and transformed into E. coli . Approximately 60 single colonies from each clone were picked at random, and the DNAs were isolated and digested with restriction enzymes to release the FRA16B insert. Samples were then electrophoresed in agarose gels to compare to unreplicated DNA and scored for insertion or deletion events (black bars). As a control, DNAs that were not transfected into HEK293T cells were also transformed into E. coli and scored for background instability in E. coli (white bars). The fractions within each bar show the number of molecules mutated over the total number of molecules examined. FRA16B instability is displayed as a percentage, and the schematic drawings above each data set show the relative location of the SV40 ori (open circles) and the replication orientation (arrows) of the FRA16B sequence (black boxes). Clone 17 demonstrates a statistically significant increase in instability events in HEK293T cells relative to background instability (* P = 0.022), and to the control (** P = 0.016). ( F ) Analysis of mutations generated by replication of clone 17 with SV40 ori in HEK293T cells. For the five mutants, the type of mutation, size of the mutation (in bp) and distance (in bp) from SV40 ori are reported in the table.
    Figure Legend Snippet: Analysis of FRA16B replication efficiency and instability in human cells using an SV40 replication system. ( A ) Diagram of replication constructs used for replication efficiency and instability assays. The bidirectional SV40 ori (open circles) was cloned into pGEM3zf(+) to generate pGEM-SV40 ori , used as a control. The 921-bp FRA16B fragment (shaded box) was inserted 30, 300, 400 or 700 bp from the SV40 ori , and in one of two replication orientations (represented by arrows). ( B ) Schematic of strategy used to determine the replication efficiency of FRA16B constructs and instability events following replication in HEK293T cells. To determine replication efficiency, equal amounts of pGEM-SV40 ori and each FRA16B-containing construct were co-transfected into HEK293T cells. After replication for 48 h, SV40 DNAs were extracted, purified and digested with DpnI to remove any unreplicated templates. Replicated molecules were then subjected to Southern blot analysis. To examine FRA16B-associated instability, each FRA16B-containing construct was transfected and replicated in HEK293T cells. SV40 DNA was extracted, purified and digested with DpnI to remove any unreplicated templates. These DNAs were transformed into E. coli , isolated, and digested with restriction enzymes to release the FRA16B insert. Replication products were analyzed on 1.5% agarose gels to score insertions and deletions. As a control, replication constructs were directly transformed into E. coli , without replication in human cells, to account for background instability in E. coli . ( C ) Replication efficiency of FRA16B constructs in HEK293T cells. The replication efficiency of each FRA16B-containing construct was determined by the ratio of replicated FRA16B DNA to the control (pGEM-SV40 ori ). Results are shown as the mean ± SD from six individual experiments. ( D ) Identification of mutation events of replication in HEK293T cells using gel electrophoresis. A representative agarose gel shows individual HEK293T cell replication products from clone 17. Replication products (#4 and #8) exhibit large base pair deletions, as demonstrated by faster migrating products (asterisks) compared to unreplicated DNA (designated as C). M, molecular weight marker. ( E ) Mutation analysis of FRA16B constructs with different replication orientations and distances from the SV40 ori . After replication in HEK293T cells, purified SV40 DNA was digested to eliminate any unreplicated parental templates and transformed into E. coli . Approximately 60 single colonies from each clone were picked at random, and the DNAs were isolated and digested with restriction enzymes to release the FRA16B insert. Samples were then electrophoresed in agarose gels to compare to unreplicated DNA and scored for insertion or deletion events (black bars). As a control, DNAs that were not transfected into HEK293T cells were also transformed into E. coli and scored for background instability in E. coli (white bars). The fractions within each bar show the number of molecules mutated over the total number of molecules examined. FRA16B instability is displayed as a percentage, and the schematic drawings above each data set show the relative location of the SV40 ori (open circles) and the replication orientation (arrows) of the FRA16B sequence (black boxes). Clone 17 demonstrates a statistically significant increase in instability events in HEK293T cells relative to background instability (* P = 0.022), and to the control (** P = 0.016). ( F ) Analysis of mutations generated by replication of clone 17 with SV40 ori in HEK293T cells. For the five mutants, the type of mutation, size of the mutation (in bp) and distance (in bp) from SV40 ori are reported in the table.

    Techniques Used: Construct, Clone Assay, Transfection, Purification, Southern Blot, Transformation Assay, Isolation, Mutagenesis, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Sequencing, Generated

    3) Product Images from "DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION"

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    Journal: Virology

    doi: 10.1016/j.virol.2009.12.026

    Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of DNA replication measured by qPCR are reported in pg of DpnI-resistant plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.
    Figure Legend Snippet: Correlation between ori-plasmid replication and firefly luciferase expression (A) Levels of SV40 plasmid replication measured by quantitative real-time PCR (qPCR) or determined by measuring levels of firefly luciferase activity. C33A cells were transfected with the pFLORI40 plasmid and the LT expression vector (+LT). As controls, cells were transfected without the LT expression vector (No LT) or with a similar plasmid lacking the ori (No ori). The amounts of DNA replication measured by qPCR are reported in pg of DpnI-resistant plasmid per mg of total genomic DNA. Firefly luciferase (Fluc) activity is reported in relative light units (RLU). (B) A similar analysis was performed for HPV31 DNA replication with the exception that cells were transfected with the pFLORI31 plasmid, or a similar plasmid lacking the ori (No ori), and with expression vectors for E1 and E2, as indicated.

    Techniques Used: Plasmid Preparation, Luciferase, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Transfection

    4) Product Images from "Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿"

    Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02651-06

    P-Cell separation of replication proteins from Ad-infected cells. (A) Western blots of fractions I, IIA, IIB, and IIC with Pol δ, Pol ɛ, Pol α, Ad DBP, RFC, RPA, and PCNA antibodies. (B) Reconstitution of AAV DNA replication in vitro using fractionated Ad-infected-cell extracts. Standard replication reaction mixtures (30 μl) contained either crude Ad-infected-cell extract (C; 200 μg) or P-Cell fractions (I, 160 μg; IIA, 36 μg; IIC′, 30 μg; IID, 9 μg) and Rep78 (3.4 μg), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. The DNA products from each reaction were digested with DpnI and analyzed on 0.8% Tris-borate-EDTA (TBE)-agarose. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion. DNA products sensitive to DpnI digestion are denoted by the black line. (C) Optimization of AAV DNA replication with RFC and P-Cell fractions I and IIA. Standard replication reaction mixtures (15 μl) contained either crude Ad-infected-cell extract (C; 100 μg) or P-Cell fraction I (80 μg), RFC (0.5 μg), Rep78 (1.7 μg), and various amounts of fraction IIA (IIA total protein concentration, 6 mg/ml), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. Half of each reaction mixture was subjected to DpnI digestion and analyzed on 0.8% TBE-agarose; the remaining half was used to quantify [ 32 P]dAMP incorporation using a DE-81 filter binding assay (expressed as pmol of dAMP incorporated per 15 μl reaction mixture). The migration pattern and fragment size of radiolabeled HindIII-digested lambda DNA are indicated on the left.
    Figure Legend Snippet: P-Cell separation of replication proteins from Ad-infected cells. (A) Western blots of fractions I, IIA, IIB, and IIC with Pol δ, Pol ɛ, Pol α, Ad DBP, RFC, RPA, and PCNA antibodies. (B) Reconstitution of AAV DNA replication in vitro using fractionated Ad-infected-cell extracts. Standard replication reaction mixtures (30 μl) contained either crude Ad-infected-cell extract (C; 200 μg) or P-Cell fractions (I, 160 μg; IIA, 36 μg; IIC′, 30 μg; IID, 9 μg) and Rep78 (3.4 μg), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. The DNA products from each reaction were digested with DpnI and analyzed on 0.8% Tris-borate-EDTA (TBE)-agarose. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion. DNA products sensitive to DpnI digestion are denoted by the black line. (C) Optimization of AAV DNA replication with RFC and P-Cell fractions I and IIA. Standard replication reaction mixtures (15 μl) contained either crude Ad-infected-cell extract (C; 100 μg) or P-Cell fraction I (80 μg), RFC (0.5 μg), Rep78 (1.7 μg), and various amounts of fraction IIA (IIA total protein concentration, 6 mg/ml), as indicated. Reaction mixtures were incubated for 2 h at 37°C, processed, and analyzed as described in Materials and Methods. Half of each reaction mixture was subjected to DpnI digestion and analyzed on 0.8% TBE-agarose; the remaining half was used to quantify [ 32 P]dAMP incorporation using a DE-81 filter binding assay (expressed as pmol of dAMP incorporated per 15 μl reaction mixture). The migration pattern and fragment size of radiolabeled HindIII-digested lambda DNA are indicated on the left.

    Techniques Used: Infection, Western Blot, Recombinase Polymerase Amplification, In Vitro, Incubation, Protein Concentration, Filter-binding Assay, Migration, Lambda DNA Preparation

    DEAE-Sepharose fractionation of DNA polymerases from P-Cell fraction IIA of uninfected-293 cell extract. (A) DNA polymerase activity of fractions from DEAE Sepharose (see Materials and Methods). (B) Western blot of DEAE fractions using anti-Pol δ and anti-Pol ɛ antibodies which were probed for sequentially without stripping the blot between antibodies. (C) In vitro AAV DNA replication. Standard replication reaction mixtures (15 μl) contained, as shown, the following components: crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), DEAE-Sepharose fractions 10 and 11 of the Pol δ pool (0.44 mg/ml), and fractions 13 to 15 of the Pol ɛ pool (2.6 mg/ml), as indicated. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion.
    Figure Legend Snippet: DEAE-Sepharose fractionation of DNA polymerases from P-Cell fraction IIA of uninfected-293 cell extract. (A) DNA polymerase activity of fractions from DEAE Sepharose (see Materials and Methods). (B) Western blot of DEAE fractions using anti-Pol δ and anti-Pol ɛ antibodies which were probed for sequentially without stripping the blot between antibodies. (C) In vitro AAV DNA replication. Standard replication reaction mixtures (15 μl) contained, as shown, the following components: crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), DEAE-Sepharose fractions 10 and 11 of the Pol δ pool (0.44 mg/ml), and fractions 13 to 15 of the Pol ɛ pool (2.6 mg/ml), as indicated. md and dd indicate monomer duplex and dimer duplex DNA species, respectively, which are resistant to DpnI digestion.

    Techniques Used: Fractionation, Activity Assay, Western Blot, Stripping Membranes, In Vitro, IA

    5) Product Images from "Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay"

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973396

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    6) Product Images from "Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay"

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973396

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    7) Product Images from "Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism"

    Article Title: Phase variation controls expression of Salmonella lipopolysaccharide modification genes by a DNA methylation-dependent mechanism

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2010.07203.x

    The GATC pairs in the gtr P22 regulatory region are differentially methylated in phase ON and OFF cells. Southern blot of chromosomal DNA probed with a gtr P22 regulatory region probe. DNA was digested with MslI and with MboI, DpnI or Sau3AI, as indicated. Control indicates DNA was digested only with MslI. A. Genomic DNA was analysed from cultures with predominantly either cells in the Lac+ (lanes 1–4) or Lac− phase of sMV83 (lanes 5–8). B. DNA analysed from sMV136 ( oxyR - ), sMV175 (−10 and −35 only), sMV174 [OxyR(BC) site only] and sMV244 [ oxyR - , OxyR(BC) site only]. C. and D. show the expected band sizes resulting from different digestions of the full length and shorter promoter constructs respectively.
    Figure Legend Snippet: The GATC pairs in the gtr P22 regulatory region are differentially methylated in phase ON and OFF cells. Southern blot of chromosomal DNA probed with a gtr P22 regulatory region probe. DNA was digested with MslI and with MboI, DpnI or Sau3AI, as indicated. Control indicates DNA was digested only with MslI. A. Genomic DNA was analysed from cultures with predominantly either cells in the Lac+ (lanes 1–4) or Lac− phase of sMV83 (lanes 5–8). B. DNA analysed from sMV136 ( oxyR - ), sMV175 (−10 and −35 only), sMV174 [OxyR(BC) site only] and sMV244 [ oxyR - , OxyR(BC) site only]. C. and D. show the expected band sizes resulting from different digestions of the full length and shorter promoter constructs respectively.

    Techniques Used: Methylation, Southern Blot, Construct

    8) Product Images from "Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance"

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance

    Journal: Journal of Virology

    doi: 10.1128/JVI.02158-18

    Deletions within LANA residues 33 to 273 do not reduce DNA replication. LANA or each LANA mutant was cotransfected with a plasmid containing 8 TR copies (p8TR-gB) into 293T cells. Twenty-four or 72 h after transfection, DNA was extracted by the method of Hirt. (A, B, D, and E) HindIII-digested Hirt DNA harvested at 24 h (A and D) or Hirt DNA digested with HindIII and DpnI at 72 h (B and E) was assessed by Southern blotting after incubation with 32 P-radiolabeled TR probe. (C and F) Western blotting for LANA or tubulin 24 h after 293T cell transfection. Brightness and contrast were uniformly adjusted using Adobe Photoshop. The results are representative of at least 3 independent experiments. The arrows in panels A and D indicate linear p8TR-gB plasmid; the arrows in panels B and E indicate replicated DNA.
    Figure Legend Snippet: Deletions within LANA residues 33 to 273 do not reduce DNA replication. LANA or each LANA mutant was cotransfected with a plasmid containing 8 TR copies (p8TR-gB) into 293T cells. Twenty-four or 72 h after transfection, DNA was extracted by the method of Hirt. (A, B, D, and E) HindIII-digested Hirt DNA harvested at 24 h (A and D) or Hirt DNA digested with HindIII and DpnI at 72 h (B and E) was assessed by Southern blotting after incubation with 32 P-radiolabeled TR probe. (C and F) Western blotting for LANA or tubulin 24 h after 293T cell transfection. Brightness and contrast were uniformly adjusted using Adobe Photoshop. The results are representative of at least 3 independent experiments. The arrows in panels A and D indicate linear p8TR-gB plasmid; the arrows in panels B and E indicate replicated DNA.

    Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Southern Blot, Incubation, Western Blot

    9) Product Images from "Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿"

    Article Title: Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02797-06

    Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.
    Figure Legend Snippet: Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.

    Techniques Used: Southern Blot, Molecular Weight, Infection, Methylation, CpG Methylation Assay

    10) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    11) Product Images from "Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts"

    Article Title: Biochemical reconstitution of abasic DNA lesion replication in Xenopus extracts

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm552

    Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). ( A ) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32 P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. ( B ) Transformation efficiency of pET28a (kan R ; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (amp R ; expressed in absolute colony numbers). ( C ) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. ( D ) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.
    Figure Legend Snippet: Determination of the nucleotides opposite the AP site in the final replication products (after 75 min of incubation in NPE). ( A ) Restriction digestion of the gel-purified supercoiled final replication products (detected by 32 P) and the control pET28a DNA (detected by SYBR Gold). R: relaxed; L: linear; S: supercoiled. ( B ) Transformation efficiency of pET28a (kan R ; expressed in percentages of the number of transformants of the uncut DNA) and AP DNA (amp R ; expressed in absolute colony numbers). ( C ) The nucleotides found at the position opposite the AP lesion in the plasmids isolated from the transformants of the DpnI and ClaI-digested Δ:C and Δ:G replication products. ( D ) The average ratios and absolute deviations of each nucleotide inserted at the position opposite the AP lesion for the Δ:C and Δ:G replication products. The data were from two independent experiments for each substrate. The right-most column listed the expected ratios if the AP lesion was replicated by random insertion of the 4 nt.

    Techniques Used: Incubation, Purification, Transformation Assay, Isolation

    Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). ( A ) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. ( B ) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. ( C ) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.
    Figure Legend Snippet: Analysis of the replication products that still carried the AP lesion (after 75 min of incubation in NPE). ( A ) Six potential types of DNA and their sensitivity to various enzymes. The lesion-carrying DNA would be nicked on the AP strand but intact on the complementary strand. BER: base excision repair; H: non-G; D: non-C. ( B ) Sequence analysis of the cloned PCR products amplified from Δ:G replication products that had been digested with DpnI, ClaI, APE and KpnI. ( C ) Sequence analysis of the cloned PCR products amplified from Δ:T replication products that had been digested with DpnI, ClaI, APE and KpnI.

    Techniques Used: Incubation, Sequencing, Clone Assay, Polymerase Chain Reaction, Amplification

    12) Product Images from "A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA ProteinsMolecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANACrystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency"

    Article Title: A Structural Basis for BRD2/4-Mediated Host Chromatin Interaction and Oligomer Assembly of Kaposi Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus LANA ProteinsMolecular Basis for Oligomeric-DNA Binding and Episome Maintenance by KSHV LANACrystal Structure of the Gamma-2 Herpesvirus LANA DNA Binding Domain Identifies Charged Surface Residues Which Impact Viral Latency

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003640

    Role of the ‘Basic top’ of LANA in BET Protein Interaction and Oligomerization. A: Co-IP of kLANA ‘basic top’ mutants with GFP-BRD4 (left) and GFP-BRD2 (right). Panels I–IV as in Figure 4B . ‘09A/38A’: K1109A/K1138A mutant. B: Oligomerization assay with kLANA ‘basic top’ mutants. Upper top: WB detecting FL kLANA wt or mutants bound to GST-kLANA wt or mutant CTDs. Lower top: same WB membrane as above detecting GST-LANA CTD proteins. (e.v.) empty vector, (-) GST alone, (MUT) mutant GST-LANA CTD corresponding to the FL LANA mutant indicated above. Bottom: Expression of FL LANA proteins and the actin control. C: EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) ‘basic top’ mutants. (wt+comp.) control with 10× excess of unlabeled probe. Below: Expression of the GST-LANA CTD proteins (Coomassie stain). D: Transient replication assay with kLANA ‘basic top’ mutants and pTR1 vector in HeLa cells (performed in duplicates). Panel I: Southern blot of replicated DNA, remaining after digest with KpnI and DpnI. Panel II: Southern blot of input DNA linearized with KpnI; pBluescript (pBS) does not replicate (internal control). (-) empty vector control. Panel III: LANA expression. Panel IV: Actin control. E: Top view of kLANA and mLANA CTDs. Mutated residues are labeled. F: Co-IP of GFP-BRD4 (left) and GFP-BRD2 (right) with HA-mLANA wt or 4A mutant. Panels I: Immunoblot of co-IP samples detecting GFP-BET proteins. Panels II: Blot of the same samples detecting HA-mLANA. Panels III: Expression of BET proteins. Panels IV: Actin loading control. G: C57BL/6 mice were infected i.n. with the MHV68 wt, the mLANA: 4A (‘4A’) and STOP (‘73-STOP’) mutant viruses and respective revertants (‘4A rev’), (‘73-STOP rev’). DNA isolated from splenocytes was used for qPCR analysis. Marks represent individual mice and the bars the means. Data include two independent experiments. (***) P
    Figure Legend Snippet: Role of the ‘Basic top’ of LANA in BET Protein Interaction and Oligomerization. A: Co-IP of kLANA ‘basic top’ mutants with GFP-BRD4 (left) and GFP-BRD2 (right). Panels I–IV as in Figure 4B . ‘09A/38A’: K1109A/K1138A mutant. B: Oligomerization assay with kLANA ‘basic top’ mutants. Upper top: WB detecting FL kLANA wt or mutants bound to GST-kLANA wt or mutant CTDs. Lower top: same WB membrane as above detecting GST-LANA CTD proteins. (e.v.) empty vector, (-) GST alone, (MUT) mutant GST-LANA CTD corresponding to the FL LANA mutant indicated above. Bottom: Expression of FL LANA proteins and the actin control. C: EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) ‘basic top’ mutants. (wt+comp.) control with 10× excess of unlabeled probe. Below: Expression of the GST-LANA CTD proteins (Coomassie stain). D: Transient replication assay with kLANA ‘basic top’ mutants and pTR1 vector in HeLa cells (performed in duplicates). Panel I: Southern blot of replicated DNA, remaining after digest with KpnI and DpnI. Panel II: Southern blot of input DNA linearized with KpnI; pBluescript (pBS) does not replicate (internal control). (-) empty vector control. Panel III: LANA expression. Panel IV: Actin control. E: Top view of kLANA and mLANA CTDs. Mutated residues are labeled. F: Co-IP of GFP-BRD4 (left) and GFP-BRD2 (right) with HA-mLANA wt or 4A mutant. Panels I: Immunoblot of co-IP samples detecting GFP-BET proteins. Panels II: Blot of the same samples detecting HA-mLANA. Panels III: Expression of BET proteins. Panels IV: Actin loading control. G: C57BL/6 mice were infected i.n. with the MHV68 wt, the mLANA: 4A (‘4A’) and STOP (‘73-STOP’) mutant viruses and respective revertants (‘4A rev’), (‘73-STOP rev’). DNA isolated from splenocytes was used for qPCR analysis. Marks represent individual mice and the bars the means. Data include two independent experiments. (***) P

    Techniques Used: Co-Immunoprecipitation Assay, Mutagenesis, Western Blot, Plasmid Preparation, Expressing, Staining, Southern Blot, Labeling, Mouse Assay, Infection, Isolation, Real-time Polymerase Chain Reaction

    Oligomerization of the KSHV and MHV-68 LANA CTDs. A : Oligomeric assemblies of kLANA (top) and mLANA (below) CTD dimers as found in the respective crystals. Inter-chain contact areas within and between dimers are indicated. B : Details on the oligomerization sites of kLANA (left) and mLANA CTDs (right), viewed from the center of the ring (kLANA, monoclinic crystal) and the top of the linear chain (mLANA). Color scheme corresponds to Figure 1A . C : Flow profiles (black graph) and molecular weight (red graph) of kLANA(1013-1149) (top) and mLANA(124-260) (bottom) in asymmetric field flow fractionation. D : Oligomerization assay with kLANA mutants. Top left: Western blot detecting FL kLANA wt or mutants bound to GST-fused kLANA wt or mutant CTDs. Bottom left: Ponceau S –stained WB membrane showing GST-LANA(934–1162) used in this assay. (e.v.) empty vector, (-) GST only, (MUT) mutant GST-LANA CTD always corresponding to the FL LANA mutant indicated above. Right: Expression of FL LANA proteins in eukaryotic cells. The aberrant running behavior of some mutants might be due to different posttranslational modification. E : EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) oligomerization deficient mutants. (wt+comp.) control with 10× excess of unlabeled probe. Right: Expression of GST-LANA CTD proteins; Coomassie stained SDS PAGE gel. F : Transient replication assay with oligomerization mutants and pGTR4 vector in HeLa cells. Panel I: Southern blot of replicated DNA, remaining after digest with MfeI and DpnI. Panel II: Southern blot of input DNA linearized with MfeI; pEGFP does not replicate and serves as an internal control. Assay was performed in duplicates. Panel III: LANA expression. Panel IV: Actin loading control. (-) empty vector control.
    Figure Legend Snippet: Oligomerization of the KSHV and MHV-68 LANA CTDs. A : Oligomeric assemblies of kLANA (top) and mLANA (below) CTD dimers as found in the respective crystals. Inter-chain contact areas within and between dimers are indicated. B : Details on the oligomerization sites of kLANA (left) and mLANA CTDs (right), viewed from the center of the ring (kLANA, monoclinic crystal) and the top of the linear chain (mLANA). Color scheme corresponds to Figure 1A . C : Flow profiles (black graph) and molecular weight (red graph) of kLANA(1013-1149) (top) and mLANA(124-260) (bottom) in asymmetric field flow fractionation. D : Oligomerization assay with kLANA mutants. Top left: Western blot detecting FL kLANA wt or mutants bound to GST-fused kLANA wt or mutant CTDs. Bottom left: Ponceau S –stained WB membrane showing GST-LANA(934–1162) used in this assay. (e.v.) empty vector, (-) GST only, (MUT) mutant GST-LANA CTD always corresponding to the FL LANA mutant indicated above. Right: Expression of FL LANA proteins in eukaryotic cells. The aberrant running behavior of some mutants might be due to different posttranslational modification. E : EMSA with LBS1+2 oligonucleotide and GST-LANA(934-1162) oligomerization deficient mutants. (wt+comp.) control with 10× excess of unlabeled probe. Right: Expression of GST-LANA CTD proteins; Coomassie stained SDS PAGE gel. F : Transient replication assay with oligomerization mutants and pGTR4 vector in HeLa cells. Panel I: Southern blot of replicated DNA, remaining after digest with MfeI and DpnI. Panel II: Southern blot of input DNA linearized with MfeI; pEGFP does not replicate and serves as an internal control. Assay was performed in duplicates. Panel III: LANA expression. Panel IV: Actin loading control. (-) empty vector control.

    Techniques Used: Flow Cytometry, Molecular Weight, Field Flow Fractionation, Western Blot, Mutagenesis, Staining, Plasmid Preparation, Expressing, Modification, SDS Page, Southern Blot, Control Assay

    13) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    14) Product Images from "An ac34 Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence In Vivo"

    Article Title: An ac34 Deletion Mutant of Autographa californica Nucleopolyhedrovirus Exhibits Delayed Late Gene Expression and a Lack of Virulence In Vivo

    Journal: Journal of Virology

    doi: 10.1128/JVI.00779-12

    qPCR analysis of viral DNA replication. Sf9 cells were transfected with 0.5 μg of vAcWT or vAc34KO bacmid DNA. At various time points, total cellular DNA was extracted, digested with DpnI, and analyzed with qPCR. The values presented for each
    Figure Legend Snippet: qPCR analysis of viral DNA replication. Sf9 cells were transfected with 0.5 μg of vAcWT or vAc34KO bacmid DNA. At various time points, total cellular DNA was extracted, digested with DpnI, and analyzed with qPCR. The values presented for each

    Techniques Used: Real-time Polymerase Chain Reaction, Transfection

    15) Product Images from "Serine Phosphoacceptor Sites within the Core Protein of Hepatitis B Virus Contribute to Genome Replication Pleiotropically"

    Article Title: Serine Phosphoacceptor Sites within the Core Protein of Hepatitis B Virus Contribute to Genome Replication Pleiotropically

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0017202

    Southern blot analysis of capsid-derived DNA isolated from transfected cells. DNA was isolated from transfected cells by two different procedures. Cytoplasmic lysates were treated with MNase to digest unencapsidated nucleic acids and capsid DNA was subsequently isolated or total cytoplasmic DNA was isolated and then treated with the restriction enzyme DpnI, which digests transfected plasmid DNA. The positions of ssDNA, dlDNA, and rcDNA are indicated and the position of a linear, 3182 bp marker is also indicated. Each lane represents 1/3 of the HBV DNA from a 60 mm dish of transfected HepG2 cells. DNA was detected by hybridizing oligonucleotide probes 1833+, 1857+, 1876+, and 1995+ ( Table S1 ). (***) indicates the discrete, incomplete rcDNA (ircDNA) molecule (See Fig. 3 ).
    Figure Legend Snippet: Southern blot analysis of capsid-derived DNA isolated from transfected cells. DNA was isolated from transfected cells by two different procedures. Cytoplasmic lysates were treated with MNase to digest unencapsidated nucleic acids and capsid DNA was subsequently isolated or total cytoplasmic DNA was isolated and then treated with the restriction enzyme DpnI, which digests transfected plasmid DNA. The positions of ssDNA, dlDNA, and rcDNA are indicated and the position of a linear, 3182 bp marker is also indicated. Each lane represents 1/3 of the HBV DNA from a 60 mm dish of transfected HepG2 cells. DNA was detected by hybridizing oligonucleotide probes 1833+, 1857+, 1876+, and 1995+ ( Table S1 ). (***) indicates the discrete, incomplete rcDNA (ircDNA) molecule (See Fig. 3 ).

    Techniques Used: Southern Blot, Derivative Assay, Isolation, Transfection, Plasmid Preparation, Marker

    16) Product Images from "Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay"

    Article Title: Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

    Journal: bioRxiv

    doi: 10.1101/2020.03.02.973396

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).
    Figure Legend Snippet: Multiplexed Integrated Accessibility Assay (MIAA) measures local DNA accessibility of synthesized oligonucleotide phrase libraries. A) 100nt phrases are integrated into stem cells at a designated genomic locus. Stem cells are split and half are differentiated into definitive endoderm cells. Retinoic acid receptor fused to hyper-activated deoxyadenosine methylase (DAM) enzyme results in methylation of phrases that open DNA. DNA is extracted and half is exposed to DpnII, which cleaves unmethylated sequences, while half is exposed to DpnI, which cleaves methylated sequences. Sequences are PCR amplified and sequenced. B) DpnI and DpnII read counts measured from a single definitive endoderm replicate show difference between designed opening and closing phrases. C) Proportion of DpnII read counts measured from a single definitive endoderm replicate gives estimate of MIAA openness. D) 100nt native sequences that were differentially opening as measured by DNase-seq are differentially opening as measured by MIAA and different from randomly shuffled control sequences (significance measured by paired t-test). E) Differential accessibility as measured by log change in normalized DNase-seq reads and MIAA methylation proportion shows correlation between native differential accessibility and MIAA accessibility. The correlation reported is the Pearson correlation coefficient (r).

    Techniques Used: Synthesized, Methylation, Polymerase Chain Reaction, Amplification

    17) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    18) Product Images from "OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica"

    Article Title: OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1483

    Methylation state of GATC sites in the opvAB regulatory region in wild-type and oxyR backgrounds. ( A ) Southern blot of genomic DNA obtained from wild-type and oxyR cultures and digested with HaeIII and with AccI (control) and DpnI, MboI or Sau3AI. Fragment sizes are indicated in base pairs. ( B ) Diagram of the HaeIII-AccI fragment and pattern of fragments obtained.
    Figure Legend Snippet: Methylation state of GATC sites in the opvAB regulatory region in wild-type and oxyR backgrounds. ( A ) Southern blot of genomic DNA obtained from wild-type and oxyR cultures and digested with HaeIII and with AccI (control) and DpnI, MboI or Sau3AI. Fragment sizes are indicated in base pairs. ( B ) Diagram of the HaeIII-AccI fragment and pattern of fragments obtained.

    Techniques Used: Methylation, Southern Blot

    19) Product Images from "Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation ▿"

    Article Title: Autographa californica Multiple Nucleopolyhedrovirus Ac92 (ORF92, P33) Is Required for Budded Virus Production and Multiply Enveloped Occlusion-Derived Virus Formation ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01598-10

    Viral DNA replication. Cells were transfected with bacmid DNA in triplicate. At 0, 24, 48, and 72 h p.t., total intracellular DNA was purified, digested with DpnI, and analyzed by real-time PCR. Two-way analysis of variance was carried out with GraphPad
    Figure Legend Snippet: Viral DNA replication. Cells were transfected with bacmid DNA in triplicate. At 0, 24, 48, and 72 h p.t., total intracellular DNA was purified, digested with DpnI, and analyzed by real-time PCR. Two-way analysis of variance was carried out with GraphPad

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

    20) Product Images from "CAG?CTG repeat instability in cultured human astrocytes"

    Article Title: CAG?CTG repeat instability in cultured human astrocytes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl614

    Analysis of SVG-A cells and transfected TNR plasmid DNA. ( A ) SVG-A cells stained with anti-GFAP antibodies (green false color) and counterstained with DAPI (blue false color) at 400× magnification. ( B ) DpnI resistance assay after recovery of plasmid pBL248 from SVG-A cells and subsequent PCR analysis of contracted alleles after yeast selection. The left panel shows the result of DpnI digestion of stock plasmid used for background determinations (lane 1 = uncut, lane 2 = cut) and plasmid DNA that has been passaged through SVG-A cells (lane 3 = uncut, lanes 4–6 = cut). Sample wells between lanes 2 and 3 and lanes 3 and 4 were intentionally left empty. The right panel shows the analysis of individual contraction events from SVG-A cells. PCR amplification of starting plasmid DNA indicates the size of the 33-repeat parental allele (lane 7). Individual contractions are shown in lanes 8–13. Fragment sizes, stated as repeat units, were deduced by comparison with molecular weight standards (data not shown).
    Figure Legend Snippet: Analysis of SVG-A cells and transfected TNR plasmid DNA. ( A ) SVG-A cells stained with anti-GFAP antibodies (green false color) and counterstained with DAPI (blue false color) at 400× magnification. ( B ) DpnI resistance assay after recovery of plasmid pBL248 from SVG-A cells and subsequent PCR analysis of contracted alleles after yeast selection. The left panel shows the result of DpnI digestion of stock plasmid used for background determinations (lane 1 = uncut, lane 2 = cut) and plasmid DNA that has been passaged through SVG-A cells (lane 3 = uncut, lanes 4–6 = cut). Sample wells between lanes 2 and 3 and lanes 3 and 4 were intentionally left empty. The right panel shows the analysis of individual contraction events from SVG-A cells. PCR amplification of starting plasmid DNA indicates the size of the 33-repeat parental allele (lane 7). Individual contractions are shown in lanes 8–13. Fragment sizes, stated as repeat units, were deduced by comparison with molecular weight standards (data not shown).

    Techniques Used: Transfection, Plasmid Preparation, Staining, Polymerase Chain Reaction, Selection, Amplification, Molecular Weight

    21) Product Images from "Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿"

    Article Title: Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02175-10

    RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.
    Figure Legend Snippet: RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.

    Techniques Used: Plasmid Preparation, Expressing, Southern Blot, Two Tailed Test, Western Blot, Transfection

    Insertion of a non-G-rich transcript disrupts OriLyt function. (A) Wild-type or mutant pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells with or without the BZLF1 gene. Plasmids were analyzed by Southern blotting before (data not shown) or after (as indicated) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (B) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for wild-type OriLyt. Data averages from three independent experiments are shown, with error bars representing the standard deviations. Statistical significance was determined using a two-tailed, unpaired t test.
    Figure Legend Snippet: Insertion of a non-G-rich transcript disrupts OriLyt function. (A) Wild-type or mutant pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells with or without the BZLF1 gene. Plasmids were analyzed by Southern blotting before (data not shown) or after (as indicated) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (B) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for wild-type OriLyt. Data averages from three independent experiments are shown, with error bars representing the standard deviations. Statistical significance was determined using a two-tailed, unpaired t test.

    Techniques Used: Mutagenesis, Plasmid Preparation, Southern Blot, Transfection, Two Tailed Test

    BHLF1 RNA is required for OriLyt function in cis . (A) Insertional mutations were made to disrupt several regions of the OriLyt sequence cloned into the pBluescript vector. These mutant plasmids were then tested for their ability to support lytic replication. (B) Wild-type or mutant pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells along with the BZLF1 gene. Plasmids were analyzed by Southern blotting before (top panel) or after (center and bottom panels) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (C) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for wild-type OriLyt. Data averages from three independent experiments are shown, with error bars representing the standard deviations. Statistical significance was determined using a two-tailed, unpaired t test. (D) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.
    Figure Legend Snippet: BHLF1 RNA is required for OriLyt function in cis . (A) Insertional mutations were made to disrupt several regions of the OriLyt sequence cloned into the pBluescript vector. These mutant plasmids were then tested for their ability to support lytic replication. (B) Wild-type or mutant pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells along with the BZLF1 gene. Plasmids were analyzed by Southern blotting before (top panel) or after (center and bottom panels) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (C) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for wild-type OriLyt. Data averages from three independent experiments are shown, with error bars representing the standard deviations. Statistical significance was determined using a two-tailed, unpaired t test. (D) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Mutagenesis, Southern Blot, Transfection, Two Tailed Test, Western Blot, Expressing

    The BHLF1 or BHRF1 transcript regions are required for OriLyt function. Sequences of various lengths were amplified from the BamHI H fragment of Epstein-Barr virus, cloned into a pBluescript plasmid vector, and tested for the ability to support lytic replication. (A) Diagram of the full-length (2,372-bp) region of OriLyt L and each of the various truncations experimentally tested. (B) pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells along with the BZLF1 gene. Plasmids were analyzed by Southern blotting before (left panel) or after (center and right panels) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (C) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for full-length (2,372-bp) OriLyt. Data averages from three identical, independent experiments are shown, with error bars representing the standard deviations. (D) Western blotting was used to monitor Zta and BALF2 protein expression levels in all transfected cells.
    Figure Legend Snippet: The BHLF1 or BHRF1 transcript regions are required for OriLyt function. Sequences of various lengths were amplified from the BamHI H fragment of Epstein-Barr virus, cloned into a pBluescript plasmid vector, and tested for the ability to support lytic replication. (A) Diagram of the full-length (2,372-bp) region of OriLyt L and each of the various truncations experimentally tested. (B) pBluescript-OriLyt or vector control plasmids were transected into ZKO-293 cells along with the BZLF1 gene. Plasmids were analyzed by Southern blotting before (left panel) or after (center and right panels) transfection and lytic induction. Plasmids recovered from cells were digested with or without DpnI enzyme (as indicated). One representative experiment is shown. (C) Relative plasmid amounts were quantified using quantitative Southern blotting. Results were calculated as the DpnI-digested signal divided by the input plasmid signal and normalized to the value obtained for full-length (2,372-bp) OriLyt. Data averages from three identical, independent experiments are shown, with error bars representing the standard deviations. (D) Western blotting was used to monitor Zta and BALF2 protein expression levels in all transfected cells.

    Techniques Used: Amplification, Clone Assay, Plasmid Preparation, Southern Blot, Transfection, Western Blot, Expressing

    22) Product Images from "Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence"

    Article Title: Characterization of a DNA Adenine Methyltransferase Gene of Borrelia hermsii and Its Dispensability for Murine Infection and Persistence

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155798

    Complementation of bh0463A in a dam - Escherichia coli strain results in adenine methylation of plasmid DNA. Control plasmid pAE30 (lanes 1, 3, 5, and 7) and a bh0463A complementation plasmid (pAE35; lanes 2, 4, 6, and 8) were extracted from a dam - E . coli strain and subjected to digestion with DpnI (lanes 3 and 4), MboI (lanes 5 and 6), or Sau3AI (lanes 7 and 8). DpnI, MboI, and Sau3A1 cut GATC sequences that are adenine methylated, adenine non-methylated, or cytosine non-methylated, respectively. Sizes of selected molecular weight standards (mws) of 100 bp DNA ladder (New England Biolabs) are shown on the left in base pairs.
    Figure Legend Snippet: Complementation of bh0463A in a dam - Escherichia coli strain results in adenine methylation of plasmid DNA. Control plasmid pAE30 (lanes 1, 3, 5, and 7) and a bh0463A complementation plasmid (pAE35; lanes 2, 4, 6, and 8) were extracted from a dam - E . coli strain and subjected to digestion with DpnI (lanes 3 and 4), MboI (lanes 5 and 6), or Sau3AI (lanes 7 and 8). DpnI, MboI, and Sau3A1 cut GATC sequences that are adenine methylated, adenine non-methylated, or cytosine non-methylated, respectively. Sizes of selected molecular weight standards (mws) of 100 bp DNA ladder (New England Biolabs) are shown on the left in base pairs.

    Techniques Used: Methylation, Plasmid Preparation, Molecular Weight

    Disruption of putative methyltransferase bh0463A results in unmethylated genomic DNA. Lanes 1, 3, 5, and 7 contain wild type B . hermsii DNA; Lanes 2, 4, 6, and 8 contain DNA from Bh Δ dam . DNA is either undigested (lanes 1 and 2), digested with DpnI (lanes 3 and 4), MboI (lanes 5 and 6), or Sau3AI (lanes 7 and 8). DpnI, MboI, and Sau3A1 cut GATC sequences that are adenine methylated, adenine non-methylated, or cytosine non-methylated GATC sites, respectively. Sizes of selected molecular weight standards (mws) of λ DNA Mono cut mix (New England Biolabs) are shown on the left in base pairs.
    Figure Legend Snippet: Disruption of putative methyltransferase bh0463A results in unmethylated genomic DNA. Lanes 1, 3, 5, and 7 contain wild type B . hermsii DNA; Lanes 2, 4, 6, and 8 contain DNA from Bh Δ dam . DNA is either undigested (lanes 1 and 2), digested with DpnI (lanes 3 and 4), MboI (lanes 5 and 6), or Sau3AI (lanes 7 and 8). DpnI, MboI, and Sau3A1 cut GATC sequences that are adenine methylated, adenine non-methylated, or cytosine non-methylated GATC sites, respectively. Sizes of selected molecular weight standards (mws) of λ DNA Mono cut mix (New England Biolabs) are shown on the left in base pairs.

    Techniques Used: Methylation, Molecular Weight

    23) Product Images from "Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing"

    Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

    Journal: Nature Communications

    doi: 10.1038/ncomms11301

    Detection of 6mA by DA-6mA-seq. ( a ) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. ( b ) DA-6mA-seq identifies consistent 6mA sites as reported previously 8 . ( c ) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). ( d ) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.
    Figure Legend Snippet: Detection of 6mA by DA-6mA-seq. ( a ) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. ( b ) DA-6mA-seq identifies consistent 6mA sites as reported previously 8 . ( c ) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). ( d ) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.

    Techniques Used: Methylation, Sonication

    DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes. The PAGE gel shows the formation of digested products. ( a ) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. ( b ) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1 .
    Figure Legend Snippet: DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes. The PAGE gel shows the formation of digested products. ( a ) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. ( b ) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1 .

    Techniques Used: Cleavage Assay, Methylation, Polyacrylamide Gel Electrophoresis

    24) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    25) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    26) Product Images from "Robust HIV-1 replication in the absence of integrase function"

    Article Title: Robust HIV-1 replication in the absence of integrase function

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.997023

    Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.
    Figure Legend Snippet: Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Techniques Used: Southern Blot, Binding Assay, Infection, Plasmid Preparation, Nucleic Acid Electrophoresis

    27) Product Images from "Non-Hemagglutinating Flaviviruses: Molecular Mechanisms for the Emergence of New Strains via Adaptation to European Ticks"

    Article Title: Non-Hemagglutinating Flaviviruses: Molecular Mechanisms for the Emergence of New Strains via Adaptation to European Ticks

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0007295

    Mutagenesis of the infectious clone of TBEV. The plasmid pGGVs 660–1982 H [29] that contains the partial PrM-E gene fragment between nucleotides 660–1882 of the Vs virus genome was used as a template in PCR to synthesize megaprimers E-1171 A/G (genome positions and lengths are specified). Primers used to produce megaprimers, with targeted mutations (circles) are represented by thick arrows. Subsequently the megaprimer without the other pair of primers was used to amplify plasmid pGGVs 660–1982 H (solid circular line). The produced linear newly-synthesized complementary ssDNA molecules (nicked circular dotted line) with acquired mutations were annealed during the last step of PCR, randomly producing twice-nic ked circular DNA. Parent Dam+ methylated DNA of the pGGVs 660–1982 H was removed by DpnI endonuclease digestion to facilitate clone selection.
    Figure Legend Snippet: Mutagenesis of the infectious clone of TBEV. The plasmid pGGVs 660–1982 H [29] that contains the partial PrM-E gene fragment between nucleotides 660–1882 of the Vs virus genome was used as a template in PCR to synthesize megaprimers E-1171 A/G (genome positions and lengths are specified). Primers used to produce megaprimers, with targeted mutations (circles) are represented by thick arrows. Subsequently the megaprimer without the other pair of primers was used to amplify plasmid pGGVs 660–1982 H (solid circular line). The produced linear newly-synthesized complementary ssDNA molecules (nicked circular dotted line) with acquired mutations were annealed during the last step of PCR, randomly producing twice-nic ked circular DNA. Parent Dam+ methylated DNA of the pGGVs 660–1982 H was removed by DpnI endonuclease digestion to facilitate clone selection.

    Techniques Used: Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Produced, Synthesized, Methylation, Selection

    28) Product Images from "Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields"

    Article Title: Functionally Active Fc Mutant Antibodies Recognizing Cancer Antigens Generated Rapidly at High Yields

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01112

    Schematic representation of the pipeline for generation and production of wild-type (WT) and Fc mutant IgG antibodies. (A) WT antibody construct in pVitro1-hygro-mcs. (B) Polymerase incomplete primer extension (PIPE) PCR linearization and mutagenesis of the WT construct to generate pVitro1 DNA fragments carrying the N297Q (left, fragments 1 and 4) or S239D/I332E (right, fragments 1, 2, and 4) mutations. Mutations indicated by “*”. (C) Introduction of mutations in WT constructs through mutagenic PIPE primers. (D) DpnI digestion. (E) Enzyme-independent assembly of the linear pVitro1 fragments. (F) Bacterial transformation of the assembled constructs. (G) Confirmation of the insertion of desired mutations. (H,I) Recombinant expression in Expi293F cells (H) and purification (I) of antibody WT and mutant variants.
    Figure Legend Snippet: Schematic representation of the pipeline for generation and production of wild-type (WT) and Fc mutant IgG antibodies. (A) WT antibody construct in pVitro1-hygro-mcs. (B) Polymerase incomplete primer extension (PIPE) PCR linearization and mutagenesis of the WT construct to generate pVitro1 DNA fragments carrying the N297Q (left, fragments 1 and 4) or S239D/I332E (right, fragments 1, 2, and 4) mutations. Mutations indicated by “*”. (C) Introduction of mutations in WT constructs through mutagenic PIPE primers. (D) DpnI digestion. (E) Enzyme-independent assembly of the linear pVitro1 fragments. (F) Bacterial transformation of the assembled constructs. (G) Confirmation of the insertion of desired mutations. (H,I) Recombinant expression in Expi293F cells (H) and purification (I) of antibody WT and mutant variants.

    Techniques Used: Mutagenesis, Construct, Polymerase Chain Reaction, Electroporation Bacterial Transformation, Recombinant, Expressing, Purification

    29) Product Images from "Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies"

    Article Title: Cloning Should Be Simple: Escherichia coli DH5α-Mediated Assembly of Multiple DNA Fragments with Short End Homologies

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0137466

    pUC19- lacZα assembly assay. ( A ) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the lacZα gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene ( bla ) and the origin of replication. The fragments shared 50-bp homology at both ends. ( B ) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates ( S3 Table ). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. ( C ) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.
    Figure Legend Snippet: pUC19- lacZα assembly assay. ( A ) Two fragments were PCR-amplified from the pUC19 vector to create an efficient screen for DNA assembly capability. The smaller “insert” fragment contained the coding sequence of the lacZα gene starting at position five and some downstream vector sequence. The larger “vector” fragment contained the rest of the plasmid, including the Amp resistance gene ( bla ) and the origin of replication. The fragments shared 50-bp homology at both ends. ( B ) Blue colony formation as a function of DNA concentration. Very few white colonies were observed on any of the plates ( S3 Table ). Small numbers of blue colonies present in the vector-only transformations are indicative of the small amount of contaminating circular template pUC19 used in PCR-mediated linearization of the vector and undigested during DpnI treatment. Insert-to-vector molar ratio was maintained at 5:1, and 25 μl of cells were used, corresponding to ¼ of the recommended volume. ( C ) Effect of insert-to-vector molar ratio on assembly efficiency. The vector DNA quantity was maintained at 0.5 ng. Error bars indicate standard deviation from two independent sets of experiments.

    Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Concentration Assay, Standard Deviation

    30) Product Images from "QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides"

    Article Title: QuickLib, a method for building fully synthetic plasmid libraries by seamless cloning of degenerate oligonucleotides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175146

    Circularization of linear plasmid library and removal of starting matrix. (a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.
    Figure Legend Snippet: Circularization of linear plasmid library and removal of starting matrix. (a) Time course of the circularization reaction: a mix of four enzymes (T5 exonuclease, DNA polymerase, DNA ligase and DpnI) was added to the amplified linear plasmids and incubated for one hour at 50 C. The amount of T5 exonuclease was reduced 4-fold compared to Gibson’s protocol. Circularization products (left side) are also analyzed by restriction with AvaI (right side). The band at 3.6 kb (black arrow) is only present when the plasmids are sealed. (b) DpnI is cleaving the starting matrix at 50 C while the synthetic PCR product is resistant to its action.

    Techniques Used: Plasmid Preparation, Amplification, Incubation, Polymerase Chain Reaction

    31) Product Images from "Robust HIV-1 replication in the absence of integrase function"

    Article Title: Robust HIV-1 replication in the absence of integrase function

    Journal: bioRxiv

    doi: 10.1101/2020.03.18.997023

    Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.
    Figure Legend Snippet: Southern blot restriction enzyme and probe binding schematic. DNA from HIV-1 infected cells was digested with MscI and XhoI (and DpnI to remove residual plasmid contamination) overnight. This cuts HIV-1 DNA at the indicated points, and these DNA fragments were then separated by gel electrophoresis. A P 32 radio-labelled DNA probe that spans an MscI cut site was used to detect the DNA fragments ( A-C , approximate probe binding in purple). This probe detects a 1.9kb DNA fragment that is released by all HIV-1 DNA forms ( A-C ), and also a 2.6 kb fragment released by unintegrated linear HIV-1 DNA ( A ), a 2.8 kb fragment released by 1LTR circle DNA ( B ), and a 3.4 kb fragment released by 2LTR circle DNA ( C ). Integrated HIV-1 DNA only produces the 1.9 kb fragment.

    Techniques Used: Southern Blot, Binding Assay, Infection, Plasmid Preparation, Nucleic Acid Electrophoresis

    32) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    33) Product Images from "Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins ▿"

    Article Title: Complete In Vitro Reconstitution of Adeno-Associated Virus DNA Replication Requires the Minichromosome Maintenance Complex Proteins ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.01968-07

    Level of AAV DNA replication in HEK293 cells in the presence of siRNAs. Three targets (each) for MCM2, MCM6, and Pol δ were tested in a 24-well format. Twenty picomoles of siRNA, 200 ng of pXX6 (which expresses the Ad helper functions), and 200 ng pSM620 (infectious wild-type AAV plasmid) were transfected into 293 cells. Cells were harvested at 24 h, and low-molecular-weight DNA was extracted by Hirt precipitation and quantitated by Southern blotting using a 32 P-labeled AAV DNA probe. The amount of DpnI-resistant monomer duplex DNA was quantitated by phosphorimaging analysis and compared to standard concentrations of pSM620 plasmid DNA. 1, no siRNA; 2, control siRNA (no target); 3 to 5, siRNA for Pol δ; 6 to 8, siRNA for MCM6; 9 to 11, siRNA for MCM2. The average from four independent reactions is shown; error bars indicate standard deviations.
    Figure Legend Snippet: Level of AAV DNA replication in HEK293 cells in the presence of siRNAs. Three targets (each) for MCM2, MCM6, and Pol δ were tested in a 24-well format. Twenty picomoles of siRNA, 200 ng of pXX6 (which expresses the Ad helper functions), and 200 ng pSM620 (infectious wild-type AAV plasmid) were transfected into 293 cells. Cells were harvested at 24 h, and low-molecular-weight DNA was extracted by Hirt precipitation and quantitated by Southern blotting using a 32 P-labeled AAV DNA probe. The amount of DpnI-resistant monomer duplex DNA was quantitated by phosphorimaging analysis and compared to standard concentrations of pSM620 plasmid DNA. 1, no siRNA; 2, control siRNA (no target); 3 to 5, siRNA for Pol δ; 6 to 8, siRNA for MCM6; 9 to 11, siRNA for MCM2. The average from four independent reactions is shown; error bars indicate standard deviations.

    Techniques Used: Plasmid Preparation, Transfection, Molecular Weight, Southern Blot, Labeling

    (A) Silver stain from SDS-PAGE of the purified MCM6 His-tagged complex. The fraction shown was purified by phosphocellulose, Ni-NTA agarose, and Mono Q column chromatography as described in Methods. The positions of the MCM proteins are indicated by their appropriate numbers, 2 to 7 and 10. The positions of proteins commonly associated with the MCM complex are also indicated. Only some of the MCM or associated proteins were seen by silver staining. The left lane contains protein molecular weight markers. (B) Western blots of SDS-PAGE of the Mono Q fraction shown in panel A were blotted with antibodies as indicated, using the Milipore Immobilon reagent. In most cases one blot was sequentially examined with more than one antibody when the proteins were sufficiently separated on the gel. Panel B shows the relevant portions of six separate Western blots and in each case indicates the antibodies that were used to detect MCM-related proteins. (C) In vitro DNA replication with His-tagged MCM6 Mono Q-purified MCM complex and other purified DNA replication proteins. Standard replication reactions (15 μl) were carried out, and mixtures contained, where indicated, crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), fraction IA (6 μg), Pol δ (0.4 μg), and/or the MCM complex (25 to 250 ng). Replication products were subjected to DpnI digestion prior to electrophoresis. The amount of 32 P nucleotide incorporated into monomer duplex AAV DNA was determined by phosphorimager analysis and is indicated for each lane.
    Figure Legend Snippet: (A) Silver stain from SDS-PAGE of the purified MCM6 His-tagged complex. The fraction shown was purified by phosphocellulose, Ni-NTA agarose, and Mono Q column chromatography as described in Methods. The positions of the MCM proteins are indicated by their appropriate numbers, 2 to 7 and 10. The positions of proteins commonly associated with the MCM complex are also indicated. Only some of the MCM or associated proteins were seen by silver staining. The left lane contains protein molecular weight markers. (B) Western blots of SDS-PAGE of the Mono Q fraction shown in panel A were blotted with antibodies as indicated, using the Milipore Immobilon reagent. In most cases one blot was sequentially examined with more than one antibody when the proteins were sufficiently separated on the gel. Panel B shows the relevant portions of six separate Western blots and in each case indicates the antibodies that were used to detect MCM-related proteins. (C) In vitro DNA replication with His-tagged MCM6 Mono Q-purified MCM complex and other purified DNA replication proteins. Standard replication reactions (15 μl) were carried out, and mixtures contained, where indicated, crude extract (60 μg), RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), fraction IA (6 μg), Pol δ (0.4 μg), and/or the MCM complex (25 to 250 ng). Replication products were subjected to DpnI digestion prior to electrophoresis. The amount of 32 P nucleotide incorporated into monomer duplex AAV DNA was determined by phosphorimager analysis and is indicated for each lane.

    Techniques Used: Silver Staining, SDS Page, Purification, Column Chromatography, Molecular Weight, Western Blot, In Vitro, IA, Electrophoresis

    In vitro DNA replication with partially purified fraction IA in the presence of polyclonal MCM2 antibody (α-MCM2). Standard replication reactions (15 μl) were performed and contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), Pol δ (0.4 μg), and/or MCM2 antibody (0.1 to 1.0 μg). Replication products were subjected to DpnI digestion prior to electrophoresis.
    Figure Legend Snippet: In vitro DNA replication with partially purified fraction IA in the presence of polyclonal MCM2 antibody (α-MCM2). Standard replication reactions (15 μl) were performed and contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), Pol δ (0.4 μg), and/or MCM2 antibody (0.1 to 1.0 μg). Replication products were subjected to DpnI digestion prior to electrophoresis.

    Techniques Used: In Vitro, Purification, IA, Electrophoresis

    (A) Western blot of a crude Ad-infected cell extract and the partially purified fraction IA using anti-MCM2 antibody. (B) Immunodepletion of MCM from fraction IA with MCM2 antibody. Western blot using MCM2 antibody of the supernatant (S/N) or precipitate (IP) of fraction IA in the absence of antibody (left panel) or the presence of anti-MCM2 antibody (right panel). (C) In vitro DNA replication with the mock-treated or anti-MCM2 antibody immunodepleted fraction of IA shown in panel B. The supernatant after precipitation in the absence of antibody (no Ab: IA S/N) or in the presence of anti-MCM2 antibody (α-MCM2: IA S/N) was tested, along with the precipitant without antibody added (no Ab: IA IP) or with MCM2 antibody added (α-MCM2: IA IP). Standard replication reactions were carried out, and mixtures contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), and/or Pol δ (0.4 μg). Replication products were subjected to DpnI digestion prior to electrophoresis.
    Figure Legend Snippet: (A) Western blot of a crude Ad-infected cell extract and the partially purified fraction IA using anti-MCM2 antibody. (B) Immunodepletion of MCM from fraction IA with MCM2 antibody. Western blot using MCM2 antibody of the supernatant (S/N) or precipitate (IP) of fraction IA in the absence of antibody (left panel) or the presence of anti-MCM2 antibody (right panel). (C) In vitro DNA replication with the mock-treated or anti-MCM2 antibody immunodepleted fraction of IA shown in panel B. The supernatant after precipitation in the absence of antibody (no Ab: IA S/N) or in the presence of anti-MCM2 antibody (α-MCM2: IA S/N) was tested, along with the precipitant without antibody added (no Ab: IA IP) or with MCM2 antibody added (α-MCM2: IA IP). Standard replication reactions were carried out, and mixtures contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), and/or Pol δ (0.4 μg). Replication products were subjected to DpnI digestion prior to electrophoresis.

    Techniques Used: Western Blot, Infection, Purification, IA, In Vitro, Electrophoresis

    In vitro DNA replication with purified MCM complexes. The MCM2 preparation was purified using the MCM2 His-tagged recombinant protein, and the MCM6 preparation was purified using the MCM6 His-tagged recombinant protein. Both MCM protein samples were purified by phosphocellulose and Ni-NTA chromatography as described in Methods for MCM purification. Standard replication reactions (15 μl) were carried out, and mixtures contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), and/or Pol δ (0.4 μg). The replication products were digested with DpnI prior to electrophoresis.
    Figure Legend Snippet: In vitro DNA replication with purified MCM complexes. The MCM2 preparation was purified using the MCM2 His-tagged recombinant protein, and the MCM6 preparation was purified using the MCM6 His-tagged recombinant protein. Both MCM protein samples were purified by phosphocellulose and Ni-NTA chromatography as described in Methods for MCM purification. Standard replication reactions (15 μl) were carried out, and mixtures contained, where indicated, RFC (0.01 μg), Rep78 (0.2 μg), PCNA (0.4 μg), IA (6 μg), and/or Pol δ (0.4 μg). The replication products were digested with DpnI prior to electrophoresis.

    Techniques Used: In Vitro, Purification, Recombinant, Chromatography, IA, Electrophoresis

    34) Product Images from "Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells"

    Article Title: Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

    Journal: Genes & Development

    doi: 10.1101/gad.1369805

    pFR_Luc replicates once per cell cycle with initiation localized to the GAL4-binding sites. ( A ) Schematic diagram of time course of experimental protocol. ( B,C ) HEK293 cells transfected with plasmids expressing GAL4-Orc2 ( B ) or GAL4-Cdc6 ( C ) and pFR_luc were released from a thymidine block into medium containing BrdU and harvested 0, 12, or 36 h later. Genomic and extrachromosomal DNA was isolated, digested, and loaded onto a cesium chloride gradient to separate DNA of different densities. Fractions were collected, and the amount of genomic DNA in each fraction was determined by measuring the absorbance at 260 nm (open circles); the amount of DpnI-resistant plasmid DNA was measured by quantitative PCR (filled squares). ( D ) Primer pairs used to measure the nascent-strand abundance assay on pFR_luc by real-time quantitative PCR. ( E ) Nascent-strand DNA was purified from HEK293 cells transfected with GAL4-Orc2 (dark bars) or GAL4-Cdc6 (light bars), and DNA was quantified by quantitative PCR. The results are normalized to the amount of non-origin DNA to allow comparisons between independent experiments. Data represent the average and standard deviation of two independent experiments performed in duplicate.
    Figure Legend Snippet: pFR_Luc replicates once per cell cycle with initiation localized to the GAL4-binding sites. ( A ) Schematic diagram of time course of experimental protocol. ( B,C ) HEK293 cells transfected with plasmids expressing GAL4-Orc2 ( B ) or GAL4-Cdc6 ( C ) and pFR_luc were released from a thymidine block into medium containing BrdU and harvested 0, 12, or 36 h later. Genomic and extrachromosomal DNA was isolated, digested, and loaded onto a cesium chloride gradient to separate DNA of different densities. Fractions were collected, and the amount of genomic DNA in each fraction was determined by measuring the absorbance at 260 nm (open circles); the amount of DpnI-resistant plasmid DNA was measured by quantitative PCR (filled squares). ( D ) Primer pairs used to measure the nascent-strand abundance assay on pFR_luc by real-time quantitative PCR. ( E ) Nascent-strand DNA was purified from HEK293 cells transfected with GAL4-Orc2 (dark bars) or GAL4-Cdc6 (light bars), and DNA was quantified by quantitative PCR. The results are normalized to the amount of non-origin DNA to allow comparisons between independent experiments. Data represent the average and standard deviation of two independent experiments performed in duplicate.

    Techniques Used: Binding Assay, Transfection, Expressing, Blocking Assay, Isolation, Plasmid Preparation, Real-time Polymerase Chain Reaction, Purification, Standard Deviation

    Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.
    Figure Legend Snippet: Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.

    Techniques Used: Plasmid Preparation, Binding Assay, In Vivo, Isolation, Agarose Gel Electrophoresis, Southern Blot, Marker, Transfection, Activity Assay, Luciferase

    35) Product Images from "Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells"

    Article Title: Recruitment of ORC or CDC6 to DNA is sufficient to create an artificial origin of replication in mammalian cells

    Journal: Genes & Development

    doi: 10.1101/gad.1369805

    pFR_Luc replicates once per cell cycle with initiation localized to the GAL4-binding sites. ( A ) Schematic diagram of time course of experimental protocol. ( B,C ) HEK293 cells transfected with plasmids expressing GAL4-Orc2 ( B ) or GAL4-Cdc6 ( C ) and pFR_luc were released from a thymidine block into medium containing BrdU and harvested 0, 12, or 36 h later. Genomic and extrachromosomal DNA was isolated, digested, and loaded onto a cesium chloride gradient to separate DNA of different densities. Fractions were collected, and the amount of genomic DNA in each fraction was determined by measuring the absorbance at 260 nm (open circles); the amount of DpnI-resistant plasmid DNA was measured by quantitative PCR (filled squares). ( D ) Primer pairs used to measure the nascent-strand abundance assay on pFR_luc by real-time quantitative PCR. ( E ) Nascent-strand DNA was purified from HEK293 cells transfected with GAL4-Orc2 (dark bars) or GAL4-Cdc6 (light bars), and DNA was quantified by quantitative PCR. The results are normalized to the amount of non-origin DNA to allow comparisons between independent experiments. Data represent the average and standard deviation of two independent experiments performed in duplicate.
    Figure Legend Snippet: pFR_Luc replicates once per cell cycle with initiation localized to the GAL4-binding sites. ( A ) Schematic diagram of time course of experimental protocol. ( B,C ) HEK293 cells transfected with plasmids expressing GAL4-Orc2 ( B ) or GAL4-Cdc6 ( C ) and pFR_luc were released from a thymidine block into medium containing BrdU and harvested 0, 12, or 36 h later. Genomic and extrachromosomal DNA was isolated, digested, and loaded onto a cesium chloride gradient to separate DNA of different densities. Fractions were collected, and the amount of genomic DNA in each fraction was determined by measuring the absorbance at 260 nm (open circles); the amount of DpnI-resistant plasmid DNA was measured by quantitative PCR (filled squares). ( D ) Primer pairs used to measure the nascent-strand abundance assay on pFR_luc by real-time quantitative PCR. ( E ) Nascent-strand DNA was purified from HEK293 cells transfected with GAL4-Orc2 (dark bars) or GAL4-Cdc6 (light bars), and DNA was quantified by quantitative PCR. The results are normalized to the amount of non-origin DNA to allow comparisons between independent experiments. Data represent the average and standard deviation of two independent experiments performed in duplicate.

    Techniques Used: Binding Assay, Transfection, Expressing, Blocking Assay, Isolation, Plasmid Preparation, Real-time Polymerase Chain Reaction, Purification, Standard Deviation

    Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.
    Figure Legend Snippet: Replication initiation factors fused to GAL4 stimulate replication of a plasmid containing GAL4 DNA-binding sites in vivo. ( A ) Extrachromosomal DNA was isolated from HEK293 cells cotransfected with the indicated plasmids and pFR_Luc, which contains five GAL4-binding sites (lanes 3-10 ). After digestion with DpnI and NdeI ( A ) or NdeI alone ( B ), samples were separated by agarose gel electrophoresis, and DNA was visualized by Southern blotting using a probe to the SmaI-BstEII fragment of pFR_Luc. NdeI-digested pFR_Luc was loaded in lane 1 as a size marker for linearized plasmid. In lane 2 , pFR_Luc was digested with NdeI and DpnI as a control ensuring complete digestion by DpnI. ( C ) Replication was quantified by PhosphorImaging. (R/S) The intensity of the DpnI-resistant band in A divided by the intensity of the NdeI-digested band in B . ( D ) C33a cells were transfected with the indicated plasmids and replication measured as in A . The bottom panel represents a lighter exposure of the top panel as a control showing equal amounts of transfected DNA. ( E ) The transcriptional activity of the GAL4 fusions in A were measured by a luciferase assay. (RLU) Firefly luciferase activity under control of GAL4-binding sites was normalized to Renilla luciferase under the control of a constitutively active promoter.

    Techniques Used: Plasmid Preparation, Binding Assay, In Vivo, Isolation, Agarose Gel Electrophoresis, Southern Blot, Marker, Transfection, Activity Assay, Luciferase

    36) Product Images from "Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition"

    Article Title: Genome-wide identification of structure-forming repeats as principal sites of fork collapse upon ATR inhibition

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2018.08.047

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .
    Figure Legend Snippet: CAGAGG Repeats Impede DNA synthesis (A) Schematic of in vitro Pol δHE primer-extension assay. (B) Representative images of Pol δHE reaction products. Pol δHE DNA synthesis products from ssDNA templates containing (CAGAGG) 15 , (CCTCTG) 15 , or scrambled control inserts (purine-rich or pyrimidine-rich) with increasing reaction times (3 – 15 minutes, triangle) were separated by denaturing PAGE alongside a dideoxynucleotide sequencing of the same template (TACG). Left: (CCTCTG) 15 and (CAGAGG) 15 insert-containing templates; Right: for pyrimidine-rich scrambled control. (C) Pol δHE termination probability. Termination probability, normalized by the number of nucleotides in each region, was quantified as the ratio of DNA molecules within a specific region over these plus all longer DNA molecules. (D) Effect of (CAGAGG) n repeats on plasmid DNA synthesis in cells. Left: (CAGAGG) 105 ). Right: Representative 2D gels. Plasmid transfected cells were either untreated (UT) or treated with 0.6 μM aphidicolin (APH) for 24 hours. Isolated episomal DNA was digested with DpnI, EcoRI (RI) and Eco NI (NI) and replication intermediates were resolved by 2D neutral-neutral gel electrophoresis with Southern hybridization to the indicated probe. Arrows denote the point of divergence of the double-Y structure from the simple-Y arc. (E) Replication intermediates of plasmids containing origin-distal (CAGAGG) 105 . Left: Schematic of the ori-distal vectors(2.7 kB from the origin). Right : Representative 2D gels. Experiment was carried out as described in (A), except that the purified DNAs were digested with DpnI, PpuMI, and SacII and detected with the indicated probe. (F) Schematic of replication through ori-proximal vectors and the formation of double-Y structures. Dashed red line indicates the center of the RI-NI fragment, the expected apex of the simple-Y arc. (G) Left: Schematic of replication fork barrier (RFB) index quantitation. The RFB index is the number of double Y structures (red) divided by the number present in > 1.5N simple-Y structures (blue). Right: Quantitation of the RFB index in CAGAGG) 105 .

    Techniques Used: DNA Synthesis, In Vitro, Primer Extension Assay, Polyacrylamide Gel Electrophoresis, Sequencing, Plasmid Preparation, Transfection, Isolation, Nucleic Acid Electrophoresis, Hybridization, Purification, Quantitation Assay

    37) Product Images from "Assessing the biocompatibility of click-linked DNA in Escherichia coli"

    Article Title: Assessing the biocompatibility of click-linked DNA in Escherichia coli

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks756

    ( A ) The click-oligonucleotides used for site directed mutagenesis contained a silent C to A mutation (shown in blue), that introduces a BamHI restriction site not present in the native mCherry gene. The click-linked bases are shown in red. ( B ) Assembly of the click-linked pRSET-mCherry plasmid by site directed mutagenesis, introducing a BamHI watermark. ( C ) Gel electrophoresis (0.8% agarose gel) of SDM products (expected size 3577 bp); Lane 1, 2-log DNA ladder (New England Biolabs); lane 2, pRSET-mCherry SDM with normal primers; lane 3, pRSET-mCherry SDM with normal primers followed by DpnI digestion; lane 4, pRSET-mCherry SDM using click primers; lane 5, pRSET-mCherry SDM using click primers followed by DpnI digestion; lane 6, negative control (pRSET-mCherry SDM using water instead of primers); lane 7, negative control followed by DpnI digestion; lane 8, pRSET-mCherry template plasmid.
    Figure Legend Snippet: ( A ) The click-oligonucleotides used for site directed mutagenesis contained a silent C to A mutation (shown in blue), that introduces a BamHI restriction site not present in the native mCherry gene. The click-linked bases are shown in red. ( B ) Assembly of the click-linked pRSET-mCherry plasmid by site directed mutagenesis, introducing a BamHI watermark. ( C ) Gel electrophoresis (0.8% agarose gel) of SDM products (expected size 3577 bp); Lane 1, 2-log DNA ladder (New England Biolabs); lane 2, pRSET-mCherry SDM with normal primers; lane 3, pRSET-mCherry SDM with normal primers followed by DpnI digestion; lane 4, pRSET-mCherry SDM using click primers; lane 5, pRSET-mCherry SDM using click primers followed by DpnI digestion; lane 6, negative control (pRSET-mCherry SDM using water instead of primers); lane 7, negative control followed by DpnI digestion; lane 8, pRSET-mCherry template plasmid.

    Techniques Used: Mutagenesis, Plasmid Preparation, Nucleic Acid Electrophoresis, Agarose Gel Electrophoresis, Negative Control

    38) Product Images from "Mutant T4 DNA polymerase for easy cloning and mutagenesis"

    Article Title: Mutant T4 DNA polymerase for easy cloning and mutagenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211065

    Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb vector. The plasmid (pETMCSI) [ 21 ] was linearised by PCR using as template (A) undigested and (B) Nde I- Eco RI double-digested plasmid. Digestions with E2 and DpnI were performed at 37 °C for the durations indicated and all experiments were performed in triplicate. (C) Negative control conducted with vector or insert only, following 60 minutes of E2/DpnI digestion. V1: linearized vector. V2: vector double-digested with Nde I and Eco RI prior to linearization by PCR. I: insert.
    Figure Legend Snippet: Number of colonies obtained for cloning a 1.2 kb insert into a 4.7 kb vector. The plasmid (pETMCSI) [ 21 ] was linearised by PCR using as template (A) undigested and (B) Nde I- Eco RI double-digested plasmid. Digestions with E2 and DpnI were performed at 37 °C for the durations indicated and all experiments were performed in triplicate. (C) Negative control conducted with vector or insert only, following 60 minutes of E2/DpnI digestion. V1: linearized vector. V2: vector double-digested with Nde I and Eco RI prior to linearization by PCR. I: insert.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Negative Control

    39) Product Images from "Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex"

    Article Title: Longitudinal assessment of neuronal 3D genomes in mouse prefrontal cortex

    Journal: Nature Communications

    doi: 10.1038/ncomms12743

    Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.
    Figure Legend Snippet: Chromosomal conformations tagged by TALE Gad1 Dam. ( a ) 150 kb of linear genome surrounding chromosome 2 TALE target sequence 5′-TATTGCCAAGAGAG-3′ at −1 kb position from Gad1 TSS. Dotted arc marks loop formation mapped by ‘3C' chromosome conformation capture 19 . Position of Amplicon/primer pairs 1–8 ( Supplementary Table 2 ) for DamID quantitative PCR assays from DpnII-resistant prefrontal DNA as indicated within chr.2 position 70,304,636–70,455,066. ( b ) Dam-based 3D genome mapping. TALE Gad1 Dam methylates G (m) ATC tetramers around Gad1 TALE target sequence and at chromosomal contacts and loop formations within physical proximity to target. Methylated G m ATC tetramers are selectively resistant to DpnII digest (in contrast to DpnII-sensitive non-methylated GATC). Methylated G m ATC tetramers are selectively cut by DpnI (in contrast to DpnI-resistant non-methylated GATC). DamID–PCR amplifies across DpnII-resistant G m ATC sequences and DamID-seq is based on adaptor-mediated ligation selectively at DpnI-sensitive G m ATC. DamID–PCR products are detectable for 55 kb loop (primer pair 4), corresponding to previously reported loop formation by 3C 19 and for sequences at TALE target sequence (primer pair 7) in HSV TALE Gad1 Dam-injected PFC samples PFC1, PFC2 and PFC3. The absence of DamID–PCR product in HSV Mef2c-Dam -injected PFC4, PFC5 and PFC6 is noteworthy (see also Supplementary Fig. 6 ). ( c ) DamID quantitative PCR for G m ATC quantification from prefrontal DNA, with primers within 100 kb from TALE Gad1 target sequence (see a ), after normalization to control sequence on chromosome 18. The sharp peak at position 4, corresponding to −55 kb promoter–enhancer loop 19 and peak at position 7 at TALE target sequence are noteworthy. N =3 per group. ( d ) ChIP with anti-V5 antibody to measure sequence-specific binding of TALE Gad1 Dam-V5 at Gad1 locus. Notice robust binding at TALE target sequence (position 7, see a ) but not at neighbouring positions 5, 6. N =3 per group. ( e ) Quantitative comparison of Gad1 -TSS (−55kb) Loop by gel densitometry from DamID–PCR products for TIME A and TIME B. N =4–5 mice per group. Data in c – e shown as mean±s.e.m.

    Techniques Used: Sequencing, Amplification, Real-time Polymerase Chain Reaction, Methylation, Polymerase Chain Reaction, Ligation, Injection, Chromatin Immunoprecipitation, Binding Assay, Mouse Assay

    40) Product Images from "Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication ▿"

    Article Title: Identification of a Domain of the Baculovirus Autographa californica Multiple Nucleopolyhedrovirus Single-Strand DNA-Binding Protein LEF-3 Essential for Viral DNA Replication ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.00115-10

    Viral DNA replication in bacmid-transfected Sf21 cells. Total intracellular DNA from Sf21 cells transfected with bAcP, bKO-lef3, bKO-lef3-Ac, or bKO-lef3-Cf was isolated at 24 and 48 h posttransfection. The DNA was digested with DpnI for 16 h and analyzed
    Figure Legend Snippet: Viral DNA replication in bacmid-transfected Sf21 cells. Total intracellular DNA from Sf21 cells transfected with bAcP, bKO-lef3, bKO-lef3-Ac, or bKO-lef3-Cf was isolated at 24 and 48 h posttransfection. The DNA was digested with DpnI for 16 h and analyzed

    Techniques Used: Transfection, Isolation

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Autographa californica Multiple Nucleopolyhedrovirus ac76 Is Involved in Intranuclear Microvesicle Formation ▿
    Article Snippet: .. Prior to PCR, 5 μl of total DNA from each time point was digested with 20 units of DpnI restriction enzyme (NEB) overnight in a 50-μl reaction volume to eliminate input bacmid DNA. .. Quantitative PCR (qPCR) was performed with 10 μl of the digested DNA and the Hot Start PCR Master Mix III (Chaoshi-Bio) according to the manufacturer's instructions using the primers targeting at a 100-bp region of the gp41 gene and conditions described previously ( ).

    Isolation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C. ..

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). ..

    Plasmid Preparation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

    Construct:

    Article Title: Secondary structure formation and DNA instability at fragile site FRA16B
    Article Snippet: .. To determine replication efficiency of the constructs ( B), SV40-replicated DNAs were digested with HindIII and NdeI (New England Biolabs) to linearize the plasmids, and with DpnI (New England Biolabs) to remove unreplicated parental templates. ..

    Methylation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population
    Article Snippet: .. DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA). ..

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    New England Biolabs dpni
    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial <t>gDNA</t> samples undigested (−) or digested (+) with <t>DpnI</t> . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
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    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Journal: Development (Cambridge, England)

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    doi: 10.1242/dev.139261

    Figure Lengend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Article Snippet: Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C.

    Techniques: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    Isolation of methyladenine genomic fragments using methyl-sensitive restriction enzymes, followed by PCR amplification. DNA fragments containing methylated adenines were isolated from samples of total genomic DNA of A. pisum using the methylation-site-specific restriction enzyme DpnI , adaptor ligation and the methyl-sensitive restriction enzyme DpnII . This was followed by PCR amplification as described by Steensel and Henikoff [ 59 ]. The resulting PCR products were cloned and sequenced.

    Journal: BMC Genomics

    Article Title: Adenine methylation may contribute to endosymbiont selection in a clonal aphid population

    doi: 10.1186/1471-2164-15-999

    Figure Lengend Snippet: Isolation of methyladenine genomic fragments using methyl-sensitive restriction enzymes, followed by PCR amplification. DNA fragments containing methylated adenines were isolated from samples of total genomic DNA of A. pisum using the methylation-site-specific restriction enzyme DpnI , adaptor ligation and the methyl-sensitive restriction enzyme DpnII . This was followed by PCR amplification as described by Steensel and Henikoff [ 59 ]. The resulting PCR products were cloned and sequenced.

    Article Snippet: DNA fragments containing methylated adenines were isolated from the genomic DNA using the methylation-specific restriction enzyme DpnI together with the methyl-sensitive restriction enzyme DpnII (New England Biolabs, Ipswich, MA, USA).

    Techniques: Isolation, Polymerase Chain Reaction, Amplification, Methylation, Ligation, Clone Assay

    Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.

    Journal: Molecular Biology of the Cell

    Article Title: In Vivo Association of Ku with Mammalian Origins of DNA Replication

    doi:

    Figure Lengend Snippet: Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.

    Article Snippet: Samples were digested with 0.8 U of Dpn I ( New England Biolabs ) for 45 min at 37°C in the presence of 1× NEB 4 buffer and 100 mM NaCl.

    Techniques: Activity Assay, Western Blot, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Isolation, Negative Control, In Vitro, Purification

    STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.

    Journal: American Journal of Human Genetics

    Article Title: Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    doi:

    Figure Lengend Snippet: STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.

    Article Snippet: Episomal DNAs were digested with Dpn I (New England Biolabs) to eliminate unreplicated parental templates.

    Techniques: Stripping Membranes, Transformation Assay, Cell Culture

    Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).

    Journal: American Journal of Human Genetics

    Article Title: Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    doi:

    Figure Lengend Snippet: Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).

    Article Snippet: Episomal DNAs were digested with Dpn I (New England Biolabs) to eliminate unreplicated parental templates.

    Techniques: Construct, Cotransfection