exoiii  (New England Biolabs)


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    Name:
    Exonuclease III
    Description:

    Catalog Number:
    M0206
    Price:
    252
    Category:
    DNA Modifying Enzymes
    Applications:
    DNA Manipulation
    Size:
    25000 units
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    Structured Review

    New England Biolabs exoiii
    Exonuclease III

    https://www.bioz.com/result/exoiii/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exoiii - by Bioz Stars, 2021-07
    97/100 stars

    Images

    1) Product Images from "Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure"

    Article Title: Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku150

    Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.
    Figure Legend Snippet: Nucleosome duplexes are decoupled into mononucleosomes. ( A ) EMSA of purified nucleosome duplexes containing H3(T118ph) HO and mp2-187 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S4 ). ( B ) Quantification of fraction of nucleosome duplexes (squares), nucleosome (circles) and free DNA (diamond) species for the gel in (A) versus time. Error bars are the standard deviation of three independent experiments. ( C ) EMSA of purified nucleosome duplexes and altosomes containing H3(T118ph) HO and mp2-247 after heating at 53°C for the indicated amount of time. Nucleosome duplexes convert in part to positioned and depositioned mononucleosomes as determined by MNase and ExoIII nucleosome mapping (see Supplementary Figure S5 ). ( D ) Quantification of the fraction of nucleosome duplexes (squares), altosomes (triangles), nucleosome (circles) and free DNA (diamond) species for the gel in (C) versus time. Error bars are the standard deviation of three independent experiments.

    Techniques Used: Purification, Standard Deviation

    2) Product Images from "Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase"

    Article Title: Bacteriophage Xp10 anti-termination factor p7 induces forward translocation by host RNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv586

    p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.
    Figure Legend Snippet: p7 destabilizes ECs and favours forward translocation of RNAP. ( A ) Stability of elongation complex with 11 nt-long transcript (EC11 A1long ) on A1long (see also Supplementary Figure S2 for stability of EC20 A1long ) After incubation in TB with 1M KCl in the absence (blue plot) or the presence of p7 (red plot) for the indicated periods of time, fractions of the ECs that remained on the beads after washing were measured. Data are mean and error bars are standard deviation from two independent experiments. ( B ) ExoIII footprinting of the front edge of RNAP in the EC11 A1long formed on A1long template. Superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown below the gels. Here and after, profiles are normalized to total radioactivity in each lane. ( C ) ExoIII footprinting of the front edge of RNAP and kinetics of the intrinsic transcript hydrolysis in the absence or the presence of p7, in EC14 ScTerm and EC22 ScTerm formed on scaffold ScTerm . Superimposed densitometry profiles of the ExoIII footprinting without p7 (blue) and with p7 (red) are shown below the gels. Lesser signal in 15 min lane with p7 is due to loading defect. ( D ) ExoIII footprinting of the front edge of RNAP, intrinsic hydrolysis (30 min) and pyrophosphorolysis (10 min) in the absence or presence of p7, in EC13 Sc1 formed on scaffold Sc1 . Superimposed densitometry profiles of the Exo III footprinting without p7 (blue) and with p7 (red) are shown below the gels.

    Techniques Used: Translocation Assay, Incubation, Standard Deviation, Footprinting, Radioactivity

    p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.
    Figure Legend Snippet: p7 induces more efficient rewinding of the upstream DNA duplex that results in bias towards more forward translocated complexes and destabilization of EC. ( A ) ExoIII footprinting of the front edge of RNAP in the EC16 Sc1 in the absence or presence of p7. Superimposed densitometry profiles of the lanes (normalized as above) without p7 (blue) and with p7 (red) are shown below the gel. ( B ) KMnO 4 probing of elongation complex EC16 Sc1 in the absence or presence of p7 (probing of non-template strand in EC13 Sc1 is shown as a control for comparison of opening of T22 and T33; dashed line in densitometry profile). Non-template (left panel) or template (right panel) strand of the DNA was 5′ radiolabelled. Schemes of the complexes and superimposed densitometry profiles of the lanes without p7 (blue) and with p7 (red) are shown to the side of the gels. Profiles were normalized to total radioactivity in each lane in the gel. ( C ) EMSA of different DNA substrates in the absence or presence of increasing concentration of p7 ( Escherichia coli SSB used as a positive control). ( D ) Stability of EC13 formed on scaffolds with the different state of the upstream DNA duplex; fully complementary template and non-template ( Sc1 ), carrying mismatches of different lengths just upstream of the RNA–DNA hybrid, or lacking upstream portion of the non-template strand. The schemes of the assembled complexes are shown below the graph and sequences in Supplementary Figure S1. Measured is the fraction of ECs that remained bound to beads in the absence or the presence of p7 after 30 min incubation in TB with 1 M KCl and washing. Data are mean and error bars are standard deviation from three independent experiments. ( E ) Effect of mismatch in the upstream DNA duplex on termination pattern in the absence or the presence of p7. Transcription from EC14 ScTerm with fully complementary template and non-template DNA strands and EC14 ScTerm/mm4 carrying 4 nt long mismatch just upstream of the RNA–DNA hybrid of the termination complex. The scheme of scaffold with mismatch is shown next to the gel, asterisks indicate termination positions.

    Techniques Used: Footprinting, Radioactivity, Concentration Assay, Positive Control, Incubation, Standard Deviation

    3) Product Images from "DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞"

    Article Title: DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants *DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants * S⃞

    Journal:

    doi: 10.1074/jbc.M806809200

    Verification of the ExoIII digestion method with sequence-selective G oxidation by and riboflavin. 32 P-Labeled double-stranded oligodeoxynucleotides containing four representative G sequence contexts () were treated with ( A ) or photo-activated
    Figure Legend Snippet: Verification of the ExoIII digestion method with sequence-selective G oxidation by and riboflavin. 32 P-Labeled double-stranded oligodeoxynucleotides containing four representative G sequence contexts () were treated with ( A ) or photo-activated

    Techniques Used: Sequencing, Labeling

    Related Articles

    Incubation:

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION
    Article Snippet: To measure the amount of replicated pFLORI31 or pFLORI40, 25 μl of total genomic DNA was digested with 10 units of DpnI (New England Biolabs) for 16 hrs in a final volume of 30 μl. .. The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes. .. The primers used to amplify an 80 bp-portion of the firefly luciferase gene present on the pFLORI40 (SV40 ori), pFLORI31 (HPV31 ori) or pCI-Fluc (No ori) plasmids as well as the probe used to detect the amplicon were synthesized according to unpublished sequences validated by Dr Iain Morgan (personal communication).

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: Fragmented DNA was phosphorylated at 37 °C for 30 min in a reaction consisting of 22 μl DNA, 0.5 μl T4 PNK (T4 Polynucleotide Kinase, NEB, M0201S), 2.5 μl T4 DNA Ligase Buffer with 10 mM dATP, then purified with Oligo Clean & Concentrator Kit (Zymo, D4060). .. Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Hybridization:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Therefore, treated DNA was then tested on the sensitivity to Exo III. .. Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III. .. Presence of minicircle DNA was revealed by Southern blotting by the psbA NCR probe.

    Transfection:

    Article Title: The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA
    Article Snippet: The insoluble materials containing viral replicative intermediates and most of the cellular DNA were removed by centrifugation, and the resultant supernatant containing viral cccDNA was extracted with phenol. .. To remove the transfected linear viral DNA, the nucleic acid obtained was treated with Exonuclease III (New England Biolabs) at 37°C for 1 h, followed by treatment with mung bean nuclease (New England Biolabs) at 37°C for 30 min. ..

    other:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Exo III catalyzes a stepwise removal of mononucleotides from the 3′-hydroxyl termini of linear duplex DNA.

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    New England Biolabs exonuclease iii
    Validation of the HPV31 <t>DNA</t> replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using <t>three</t> different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.
    Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    99
    New England Biolabs exonuclease iii e coli
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Exonuclease Iii E Coli, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii e coli/product/New England Biolabs
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    Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Luciferase, Plasmid Preparation, Sequencing, Expressing, Transfection

    Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Activity Assay, Mutagenesis, Transfection, Expressing, Negative Control, Western Blot

    Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Luciferase, Plasmid Preparation, Binding Assay, Transfection, Expressing, Western Blot

    Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.

    Journal: Virology

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

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Mutagenesis, Staining, SDS Page, Purification, Fluorescence, Binding Assay, Expressing, Plasmid Preparation, Western Blot

    Exo III-treated minicircular DNA revealed after PFGE and 2D-gel electrophoresis. Exo III-treated whole-DNA extract from H. triquetra was subjected to PFGE ( A ) and 2D-gel ( B ), and detected with 1.1-kb psbA NCR fragment [and psbA gene fragment in (B) only]. (A) The 6–8 kb minicircular DNA bands (APBs) were removed in the Exo III lane, while the putative 3-kb psbA minicircle band was diminished in intensity. (B) Hybridization signals of larger than 2 kb were removed after Exo III treatment as revealed in both 2D-gels detected by psbA NCR and gene fragments. Extra spots indicated by the arrows were observed in these two images when comparing with the controls. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Journal: Nucleic Acids Research

    Article Title: The replication of plastid minicircles involves rolling circle intermediates

    doi: 10.1093/nar/gkp063

    Figure Lengend Snippet: Exo III-treated minicircular DNA revealed after PFGE and 2D-gel electrophoresis. Exo III-treated whole-DNA extract from H. triquetra was subjected to PFGE ( A ) and 2D-gel ( B ), and detected with 1.1-kb psbA NCR fragment [and psbA gene fragment in (B) only]. (A) The 6–8 kb minicircular DNA bands (APBs) were removed in the Exo III lane, while the putative 3-kb psbA minicircle band was diminished in intensity. (B) Hybridization signals of larger than 2 kb were removed after Exo III treatment as revealed in both 2D-gels detected by psbA NCR and gene fragments. Extra spots indicated by the arrows were observed in these two images when comparing with the controls. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Article Snippet: Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Hybridization

    Effects of DNA ligase/Klenow fragment on minicircular DNA. Total DNA from H. triquetra was treated with T4 DNA ligase, as well as Klenow fragment followed with T4 DNA ligase, prior to Exo III digestion. Treated DNAs were resolved by PFGE and detected the minicircular DNA signals by Southern Blot. The two upper arrows indicated APBs in the untreated control lane. The two lower arrows indicated the putative monomer of psbA minicircle signals of ∼2–3 kb. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Journal: Nucleic Acids Research

    Article Title: The replication of plastid minicircles involves rolling circle intermediates

    doi: 10.1093/nar/gkp063

    Figure Lengend Snippet: Effects of DNA ligase/Klenow fragment on minicircular DNA. Total DNA from H. triquetra was treated with T4 DNA ligase, as well as Klenow fragment followed with T4 DNA ligase, prior to Exo III digestion. Treated DNAs were resolved by PFGE and detected the minicircular DNA signals by Southern Blot. The two upper arrows indicated APBs in the untreated control lane. The two lower arrows indicated the putative monomer of psbA minicircle signals of ∼2–3 kb. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Article Snippet: Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III.

    Techniques: Southern Blot

    Southern blot analysis of HBV DNA in viral or core particles. (A) HBV DNA in viral particles. HBV particles secreted into the culture medium of the cells transfected with pBS-HBV3, WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (lanes 1 to 5) were treated with 1 mg of proteinase K per ml and 1% SDS and then directly subjected to 1% agarose gel electrophoresis. The resultant DNA was blotted to the filter paper and hybridized with an HBV DNA probe. Arrowheads indicate the positions corresponding to three different forms of HBV DNA (RC, L, and SS) and the bracket shows the position of transfected plasmid DNA. (B) HBV DNA in core particles was treated as described for panel A. Lanes 1 to 5 contain the samples from pBS-HBV3-, WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The arrowhead indicates the position of the SS form of HBV DNA. The positions of the transfected plasmid and linear DNA are also indicated.

    Journal: Journal of Virology

    Article Title: The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA

    doi:

    Figure Lengend Snippet: Southern blot analysis of HBV DNA in viral or core particles. (A) HBV DNA in viral particles. HBV particles secreted into the culture medium of the cells transfected with pBS-HBV3, WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (lanes 1 to 5) were treated with 1 mg of proteinase K per ml and 1% SDS and then directly subjected to 1% agarose gel electrophoresis. The resultant DNA was blotted to the filter paper and hybridized with an HBV DNA probe. Arrowheads indicate the positions corresponding to three different forms of HBV DNA (RC, L, and SS) and the bracket shows the position of transfected plasmid DNA. (B) HBV DNA in core particles was treated as described for panel A. Lanes 1 to 5 contain the samples from pBS-HBV3-, WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The arrowhead indicates the position of the SS form of HBV DNA. The positions of the transfected plasmid and linear DNA are also indicated.

    Article Snippet: To remove the transfected linear viral DNA, the nucleic acid obtained was treated with Exonuclease III (New England Biolabs) at 37°C for 1 h, followed by treatment with mung bean nuclease (New England Biolabs) at 37°C for 30 min.

    Techniques: Southern Blot, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Techniques: Mutagenesis, Two Tailed Test