e.coli exonuclease Search Results


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  • 99
    New England Biolabs e coli exonuclease
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    E Coli Exonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 623 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATCC e coli inoculum preparation
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    E Coli Inoculum Preparation, supplied by ATCC, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Roche e coli exonuclease iii
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    E Coli Exonuclease Iii, supplied by Roche, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare e coli exonuclease
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    E Coli Exonuclease, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs e coli exonuclease iii
    <t>DNA</t> repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and <t>(iii)</t> the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.
    E Coli Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa escherichia coli exonuclease iii
    Exonuclease <t>III</t> digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by <t>Escherichia</t> coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.
    Escherichia Coli Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche e coli exonuclease iii xth protein
    Exonuclease <t>III</t> digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by <t>Escherichia</t> coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.
    E Coli Exonuclease Iii Xth Protein, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli exonuclease
    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli <t>exonuclease</t> I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.
    E Coli Exonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher e coli exonuclease iii
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 <t>DNA.</t> DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease <t>III</t> (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    E Coli Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore escherichia coli exonuclease iii
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 <t>DNA.</t> DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease <t>III</t> (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    Escherichia Coli Exonuclease Iii, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    GE Healthcare escherichia coli exonuclease i
    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 <t>DNA.</t> DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease <t>III</t> (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.
    Escherichia Coli Exonuclease I, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw274

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

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

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

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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkw274

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

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

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

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Journal: Nature protocols

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    doi: 10.1038/s41596-018-0055-0

    Figure Lengend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Article Snippet: M0293L) Lambda Exonuclease (New England BioLabs, cat.no.

    Techniques:

    DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Journal: Nucleic Acids Research

    Article Title: Linking uracil base excision repair and 5-fluorouracil toxicity in yeast

    doi: 10.1093/nar/gkj430

    Figure Lengend Snippet: DNA repair pathways implicated in 5-FU-mediated cell killing. The model is supported by the following observations: (i) a massive amount of uracil is incorporated into DNA, but the ung1 yeast are much less sensitive to 5-FU than the wild-type strain indicating that uracilated DNA is not the mediator of 5-FU toxicity; (ii) the apn1apn2ntg1ntg2 strain that is entirely defective in processing abasic sites by a BER mechanism is more sensitive to 5-FU, indicating that intact abasic sites (or repair products derived from abasic sites) have inherent toxicity; and (iii) the rad27 and apn1rad27 yeast strains show protection against 5-FU toxicity, suggesting the presence of a toxic repair intermediate downstream of the Rad27 flap endonuclease reaction. Several backup pathways for repair of abasic sites and 5′dRp groups are indicated. The lower path involving Apn2 and other BER enzymes is important in the absence of Apn1 and accounts for the efficient removal of abasic sites in the apn1 strain. NER and HR pathways are likely to be important with the apn1apn2ntg1ntg2 and rad27 knockout strains. Consistent with this, yeast deficient in both BER and NER are not viable.

    Article Snippet: Briefly, 4 µg of each DNA sample was digested with E.coli exonuclease III (145 U; New England Biolabs) for 1 min at 37°C, 100 mM putrescine at 37°C for 30 min (Acros Organics), both exonuclease III and putrescine, or left undigested.

    Techniques: Derivative Assay, Knock-Out

    The extended G-overhang is not due to the collapse of replication fork. ( A ) No 5′ C-rich overhang was detected during the cell cycle. Genomic DNA isolated from synchronized HeLa cells were hybridized to an 18-mer G-rich probe for detecting C-rich overhangs and hybridized to an 18-mer C-rich probe for detecting G-overhangs under native conditions. To determine the hybridization signals contributed by G- or C-rich overhangs, DNA was digested with 3′ → 5′ exonuclease ExoI, which specifically removes 3′ G-overhangs, or the 5′ → 3′ exonuclease RecJ f ) before hybridization. The same gels were then denatured to determine the total telomere signal. Asyn, asynchronized HeLa cells. ( B ) Quantitation of relative amount of G-overhangs from ( A ). ( C ) Hybridization of G-rich probe to (C 3 TA 2 ) 4 oligo. The ss (C 3 TA 2 ) 4 oligo was hybridized to G-rich probe under the native condition identical to ( A ) and separated in 20% native 0.5 × TBE polyacrylamide gel.

    Journal: The EMBO Journal

    Article Title: Molecular steps of G-overhang generation at human telomeres and its function in chromosome end protection

    doi: 10.1038/emboj.2010.156

    Figure Lengend Snippet: The extended G-overhang is not due to the collapse of replication fork. ( A ) No 5′ C-rich overhang was detected during the cell cycle. Genomic DNA isolated from synchronized HeLa cells were hybridized to an 18-mer G-rich probe for detecting C-rich overhangs and hybridized to an 18-mer C-rich probe for detecting G-overhangs under native conditions. To determine the hybridization signals contributed by G- or C-rich overhangs, DNA was digested with 3′ → 5′ exonuclease ExoI, which specifically removes 3′ G-overhangs, or the 5′ → 3′ exonuclease RecJ f ) before hybridization. The same gels were then denatured to determine the total telomere signal. Asyn, asynchronized HeLa cells. ( B ) Quantitation of relative amount of G-overhangs from ( A ). ( C ) Hybridization of G-rich probe to (C 3 TA 2 ) 4 oligo. The ss (C 3 TA 2 ) 4 oligo was hybridized to G-rich probe under the native condition identical to ( A ) and separated in 20% native 0.5 × TBE polyacrylamide gel.

    Article Snippet: To remove the 3′ G-rich overhang, total DNA was treated with the 3′ → 5′ exonuclease Escherichia coli ExoI (0.3 U/μg DNA, NEB) in 15 μl buffer (10 mM HEPES pH 7.5, 100 mM LiCl, 2.5 mM MgCl2 , 5 mM CaCl2 , 20 mM β-mercaptoethanol, 0.067 μg/μl DNase-free RNaseA) at 37°C for 1 h to overnight.

    Techniques: Isolation, Hybridization, Quantitation Assay

    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), 1 μl Exonuclease III (NEB, M0206S) and 1 μl Fpg (formamidopyrimidine DNA glycosylase, NEB, M0240S) were added into the reaction and jointly incubated at 37 °C for 1 h. Then the mixture was purified with MinElute Reaction Cleanup Kit (3 × ERC) (QIAGEN) and its final concentration was calibrated using Qubit ssDNA Assay Kit.

    Techniques: Mutagenesis, Two Tailed Test

    Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Journal: FEBS Open Bio

    Article Title: Contribution of histone N-terminal tails to the structure and stability of nucleosomes

    doi: 10.1016/j.fob.2013.08.007

    Figure Lengend Snippet: Exonuclease III digestion patterns of wt and tailless nucleosomes. Nucleosomes were digested for 0 (lanes 2, 6, 10, 14, and 18), 2 (lanes 3, 7, 11, 15, and 19), 4 (lanes 4, 8, 12, 16, and 20), or 8 (lanes 5, 9, 13, 17, and 21) min at 37 °C by Escherichia coli exonuclease III. The reaction was stopped by the addition of proteinase K, and the DNA was extracted with phenol/chloroform, precipitated with ethanol, and dissolved in Hi–Di Formamide. The purified DNA samples were analyzed by 10% denaturing PAGE.

    Article Snippet: Briefly, each reconstituted nucleosome, containing tlH2A, tlH2B, tlH3, or tlH4, was treated with 5 units of Escherichia coli exonuclease III (Takara), in 10 μl of 50 mM Tris–HCl (pH 8.0), 5 mM MgCl2 , and 1 mM DTT.

    Techniques: Purification, Polyacrylamide Gel Electrophoresis

    Further Characterization of ssDNA-Cohesin Interactions, Related to Figure 3 (A) ssDNA and dsDNA binding of cohesin were analyzed by electrophoretic mobility shift experiments using the indicated cohesin concentrations and single or double stranded pBluescript as the substrate. (B) ssDNA is topologically entrapped by cohesin. A schematic of the DNA-release experiment following ssDNA to dsDNA conversion is shown together with a gel image of the input and recovered DNAs at the indicated stages. (C) Cohesin releases circular ssDNA. The released DNA from cohesin following dsDNA to ssDNA conversion, as shown in Figure 3 D, was treated with E. coli exonuclease I that digests linear but not circular ssDNA. No detectable digestion was observed, suggesting that released ssDNA remained circular. As a control for the effectiveness of exonuclease I treatment, heat-denatured nicked circular DNA was treated with exonuclease I in the same way. Linear, but not circular ssDNA was readily digested under these conditions. Note that two nicks were present in the circular DNA before denaturation. This generated two linear ssDNA, only the longer one of which is visible on the gel. (D) ssDNA stimulates the cohesin ATPase. Mis4-Ssl3 and DNA-dependent ATP hydrolysis by cohesin was measured in the presence of indicated proteins and DNAs.

    Journal: Cell

    Article Title: Establishment of DNA-DNA Interactions by the Cohesin Ring

    doi: 10.1016/j.cell.2017.12.021

    Figure Lengend Snippet: Further Characterization of ssDNA-Cohesin Interactions, Related to Figure 3 (A) ssDNA and dsDNA binding of cohesin were analyzed by electrophoretic mobility shift experiments using the indicated cohesin concentrations and single or double stranded pBluescript as the substrate. (B) ssDNA is topologically entrapped by cohesin. A schematic of the DNA-release experiment following ssDNA to dsDNA conversion is shown together with a gel image of the input and recovered DNAs at the indicated stages. (C) Cohesin releases circular ssDNA. The released DNA from cohesin following dsDNA to ssDNA conversion, as shown in Figure 3 D, was treated with E. coli exonuclease I that digests linear but not circular ssDNA. No detectable digestion was observed, suggesting that released ssDNA remained circular. As a control for the effectiveness of exonuclease I treatment, heat-denatured nicked circular DNA was treated with exonuclease I in the same way. Linear, but not circular ssDNA was readily digested under these conditions. Note that two nicks were present in the circular DNA before denaturation. This generated two linear ssDNA, only the longer one of which is visible on the gel. (D) ssDNA stimulates the cohesin ATPase. Mis4-Ssl3 and DNA-dependent ATP hydrolysis by cohesin was measured in the presence of indicated proteins and DNAs.

    Article Snippet: The recovered DNA was incubated with E. coli exonuclease I (0.5 U/ μl, TaKaRa Bio) in 10 μl of exoI buffer at 30°C for 15 min.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Generated

    Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli exonuclease I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.

    Journal: Nucleic Acids Research

    Article Title: Removal of mismatched bases from synthetic genes by enzymatic mismatch cleavage

    doi: 10.1093/nar/gni058

    Figure Lengend Snippet: Principle of mismatch removal. In the first step, a double-stranded polynucleotide with a mismatching base pair, e.g. resulting from ligation of oligonucleotides in a gene synthesis reaction, is cleaved by the EMC enzyme in both strands, 2–5 bp downstream of the mismatch. The short overhangs thus generated dissociate immediately at the reaction temperature of 37°C. The resulting single-stranded overhangs can be removed by different strategies: addition of a single-strand-specific 3′–5′-exonuclease, e.g. E.coli exonuclease I in the EMC reaction or in a subsequent exonuclease step. Alternatively, the 3′–5′-exonuclease activity of proofreading polymerases can be used in the subsequent PCR. The proposed mechanism in this case is (i) removal of single-stranded overhangs during the initial heating step from 20 to 95°C or (ii) removal of mispaired bases by ‘proofreading’ in the first elongation cycle of the overlap extension PCR.

    Article Snippet: Cleavage was performed at 37°C in the buffer recommended by the manufacturer, for up to 24 h. For removal of single-stranded DNA, E.coli exonuclease I (Fermentas, St Leon-Rot, Germany) was included in some reactions.

    Techniques: Ligation, Generated, Activity Assay, Polymerase Chain Reaction

    Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Journal: Genes & Development

    Article Title: Long palindromes formed in Streptomyces by nonrecombinational intra-strand annealing

    doi:

    Figure Lengend Snippet: Southern blot of pQC110 and pQC26-derived DNAs isolated from ZX7 transformants. ( A ) from transformants receiving Dra I-cleaved pQC110 DNA. DNAs were electrophoresed for 13 hr at 40 v in 0.5% agarose gel and probed with 32 P-labled pQC110 DNA (lanes 4 – 17 ). Molecular lengths were calculated relative to Hin dIII-treated bacteriophage λ DNA (lane 1 ), a 1-kb DNA-size ladder (Life Technologies, Inc.) (lane 2 ), or covalently closed circular pQC110 DNA isolated from E. coli (lane 3 ). ( B ) Surviving replicons are linear plasmids. Lanes 4 – 7 (NT) show DNA isolated from 4 randomly selected transformants by proteinase K/SDS treatment. Aliquots of the same DNAs were treated with 100 units exonuclease III (lanes 8 – 11 ) or 10 units λ exonuclease (lanes 12 – 15 ) at 37°C for 4 hr and electrophoresed for 18 hr at 38 v in 0.5 % agarose gel. λ Hin dIII-treated DNA (lane 1 ), 1-kb DNA ladder (lane 2 ), and pQC110 DNA (from E. coli , lane 3 ) are molecular size markers. ( C ) Electrophoresis of pQC110-derived DNAs shown in A after treatment with NaOH and renaturation. Lane designations are as in A . ( D ) Bam HI digestion of pQC110-derived DNAs from A . Lane 1 contains a 1-kb ladder. Lanes 2 – 15 correspond to DNAs in lanes 4 – 17 of A . The 8.5-kb and 5-kb DNA bands discussed in the text are indicated. ( E ) Bam HI digestion of pQC110-derived DNAs following denaturation and renaturation. Lanes 2 – 16 correspond to DNAs in lanes 3 – 17 of A . ( F ) Effect of denaturation on migration of Bam HI fragments containing putative palindrome apices of linear plasmids. Agarose gel analysis of inserts recovered from agarose gel following Bam HI digestion of pQC143–pQC146. The banding position of DNAs dissolved in TE (lanes 2 – 5 ) or analyzed following treatment with NaOH and neutralization is shown in lanes 6 – 9 . ( G ) Endonuclease analysis of Bam HI fragments containing putative palindrome apices. DNAs were digested by the enzymes indicated and electrophoresed for 3 hr at 80 v on 1% agarose gel. Lanes 2, 4, 6, 8 , and lanes 10, 12, 14, 16 correspond to lanes 2 – 5 from F . Lanes 3, 5, 7, 9 , and lanes 11, 13, 15, 17 correspond to lanes 6 – 9 from F . ( H ) Effect of denaturation on migration of Sac I-cleaved pQC26 DNA isolated from four transformants by adding proteinase K/SDS (lanes 3 – 7 ) or NaOH/SDS (lanes 8 – 12 ), and electrophoresed for 20 hr at 36 v in 0.5% agarose gel. Lane 1 (1-kb ladder) and 2 (pQC26, from E. coli ) are markers.

    Article Snippet: Aliquots of DNA were incubated with 100 units of E. coli exonuclease III or 10 units of bacteriophage λ exonuclease (either purchased from Life Technologies, Inc. or a gift of Drs. Deb Chatterjee and Per Harbury) at 37°C for 1 hr and the completeness of their digestion was confirmed by gel electrophoresis.

    Techniques: Southern Blot, Derivative Assay, Isolation, Agarose Gel Electrophoresis, Electrophoresis, Migration, Neutralization