escherichia coli dna polymerase i Search Results


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    New England Biolabs escherichia coli dna polymerase i
    DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and <t>DNA-damaging</t> agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA <t>polymerase</t> I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]
    Escherichia Coli Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 256 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Thermo Fisher escherichia coli dna polymerase i
    Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli <t>DNA</t> polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.
    Escherichia Coli Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore e coli dna polymerase
    Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli <t>DNA</t> polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.
    E Coli Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs escherichia coli dna polymerase i klenow fragment
    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the <t>Klenow</t> fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of <t>hetero-DNA</t> primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).
    Escherichia Coli Dna Polymerase I Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 33 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche e coli klenow fragment
    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the <t>Klenow</t> fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of <t>hetero-DNA</t> primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).
    E Coli Klenow Fragment, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa e coli dna polymerase i
    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the <t>Klenow</t> fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of <t>hetero-DNA</t> primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).
    E Coli Dna Polymerase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim escherichia coli dna polymerase i
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    Escherichia Coli Dna Polymerase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    SibEnzyme escherichia coli dna polymerase i
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    Escherichia Coli Dna Polymerase I, supplied by SibEnzyme, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher eschericcia coli dna pol i
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    Eschericcia Coli Dna Pol I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Promega e coli dna polymerase i
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    E Coli Dna Polymerase I, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher escherichia coli dna polymerase klenow fragment i
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    Escherichia Coli Dna Polymerase Klenow Fragment I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche escherichia coli dna polymerase i klenow
    Neutralization of <t>DNA</t> polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA <t>polymerase</t> I (Klenow fragment).
    Escherichia Coli Dna Polymerase I Klenow, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore escherichia coli dna polymerase i
    Loss of E2F1 attenuates <t>DNA</t> repair. A , comet assay expressed as average tail moment on E2F1 +/+ and E2F1 -/- MEFs untreated ( left-hand graph , control), after incubation for 1 h in medium containing 50 μg/ml MMS ( middle graph, 0 h ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 2 h ( right-hand graph, R ). Experiments were performed in duplicate, and measurement of mean tail moment was from 50 cells/slide from 15-20 randomly selected fields representing the whole area of each slide. Statistical analysis was performed using unpaired two-tailed t test on comet tail moments that were determined using Comet Assay II software (Perceptive Instruments; Suffolk, UK). Error bars , S.D. from separate experiments. B , indirect immunofluorescence microscopy on XRCC1 immunostaining ( top ), DNA polymerase I-mediated FITC-dUTP labeling assay ( middle ), and 4′,6-diamidino-2-phenylindole ( DAPI ) staining ( bottom ) performed on E2F1 -/- and E2F1 +/+ MEFs untreated ( C ), after incubation for 1 h in medium containing 50 μg/ml MMS ( 0 h panels ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 6 h ( R panels ). C , percentage of apoptosis as determined by Annexin V staining and flow cytometry 48 h after infection of the indicated cells with adenovirus expressing wild-type E2F1 ( Adeno-E2F1 ) or adenovirus expressing GFP ( Adeno-control ). Error bars , S.D. of triplicate experiments and statistical analysis performed using Student's unpaired two-tailed t test.
    Escherichia Coli Dna Polymerase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 31 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]

    Journal: International Journal of Molecular Sciences

    Article Title: DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide

    doi: 10.3390/ijms150713111

    Figure Lengend Snippet: DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]

    Article Snippet: 500 μg of DNA was incubated for 40 s at 16 °C with a mixture of 200 μM of dATP, dGTP, dCTP and 20 μM of digoxigenin-11-dUTP (Roche), 117 μM of ddATP, ddGTP, ddCTP (Roche) and 1000 units of Escherichia coli DNA Polymerase I (New England Biolabs, Ipswich, MA, USA).

    Techniques: Generated, Nick Translation

    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Journal: Nucleic Acids Research

    Article Title: Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

    doi: 10.1093/nar/gkn575

    Figure Lengend Snippet: PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of DNA polymerase I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.

    Article Snippet: Primer extension with Klenow fragment of DNA polymerase Primer extension experiments using large fragment (Klenow) of DNA polymerase I (New England Biolabs) were performed at 25°C using the HIV-1 tat reverse primer (5′-AATACTATGGTCCACACAACTATTGCT-3′) that was unmodified or contained a single OXP modification.

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    DPE-PCR enables sensitive and quantitative detection of Gram-negative and Gram-positive bacteria via measurement of DPE activity in crude lysates. ( A ) Decreasing amounts of E. coli colony forming unit were spiked into bead lysis-coupled DPE-PCR. NIC were also included to monitor reagent background levels. All colony forming unit spikes and NICs were performed in triplicate. A representative DPE-PCR curve is shown below for each level of bacterial input. Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S5 ( B ) A plot of E. coli DNA polymerase activity and linear regression analysis is presented. Graphs were generated using the average C t values obtained from triplicate reactions of bacterial spikes ranging from 1 × 10 5 to 1 × 10 1 input colony forming unit. ( C and D ) Colony forming unit titration experiments were performed for S. aureus exactly as described above for E. coli . Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S6 .

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: DPE-PCR enables sensitive and quantitative detection of Gram-negative and Gram-positive bacteria via measurement of DPE activity in crude lysates. ( A ) Decreasing amounts of E. coli colony forming unit were spiked into bead lysis-coupled DPE-PCR. NIC were also included to monitor reagent background levels. All colony forming unit spikes and NICs were performed in triplicate. A representative DPE-PCR curve is shown below for each level of bacterial input. Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S5 ( B ) A plot of E. coli DNA polymerase activity and linear regression analysis is presented. Graphs were generated using the average C t values obtained from triplicate reactions of bacterial spikes ranging from 1 × 10 5 to 1 × 10 1 input colony forming unit. ( C and D ) Colony forming unit titration experiments were performed for S. aureus exactly as described above for E. coli . Colony count plating and gsPCR were performed in an effort to obtain a better estimate of the actual colony forming unit placed into each reaction and is presented in Supplementary Figure S6 .

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Activity Assay, Lysis, Generated, Titration

    Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Purification, Polymerase Chain Reaction, Construct

    Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

    DPE-PCR as an indicator of E. coli viability in response to heat treatment. ( A ) Aliquots of an E. coli suspension (∼2000 cfu/µl) were incubated at 25°C, 45°C, 65°C, 85°C and 105°C for 20 min. After heating, each bacterial stock was cooled to room temperature and 5 µl were transferred to the bead lysis-coupled DPE-PCR assay. DPE-PCR curves representing E. coli -derived DNA polymerase activity following each of the indicated temperature treatments are presented. ( B ) Plots were generated from triplicate DPE-PCRs and gsPCR of genomic DNA (from the same lysates) after the indicated temperature treatments of E. coli suspensions. Parallel plating was also performed in triplicate for each of the treated E. coli suspensions. Representative colony forming unit monitoring plates are presented below the graph, revealing bacterial viability status after treatment at each temperature. ( C ) DPE-PCR is compared with gsPCR of genomic DNA in response to the various temperature treatments. ‘Fold Reduction of qPCR Signal’ was calculated using the indicated equation and the values obtained were used to generate comparative bar graphs.

    Journal: Nucleic Acids Research

    Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

    doi: 10.1093/nar/gks316

    Figure Lengend Snippet: DPE-PCR as an indicator of E. coli viability in response to heat treatment. ( A ) Aliquots of an E. coli suspension (∼2000 cfu/µl) were incubated at 25°C, 45°C, 65°C, 85°C and 105°C for 20 min. After heating, each bacterial stock was cooled to room temperature and 5 µl were transferred to the bead lysis-coupled DPE-PCR assay. DPE-PCR curves representing E. coli -derived DNA polymerase activity following each of the indicated temperature treatments are presented. ( B ) Plots were generated from triplicate DPE-PCRs and gsPCR of genomic DNA (from the same lysates) after the indicated temperature treatments of E. coli suspensions. Parallel plating was also performed in triplicate for each of the treated E. coli suspensions. Representative colony forming unit monitoring plates are presented below the graph, revealing bacterial viability status after treatment at each temperature. ( C ) DPE-PCR is compared with gsPCR of genomic DNA in response to the various temperature treatments. ‘Fold Reduction of qPCR Signal’ was calculated using the indicated equation and the values obtained were used to generate comparative bar graphs.

    Article Snippet: DPE reaction conditions DNA Pol I (NEB cat# M0209L), Klenow (NEB cat# M0210S) and Klenow exo(−) (NEB cat# M0212S) were diluted to the indicated units per microliter stock in sterile Tris–EDTA, pH 8.0.

    Techniques: Polymerase Chain Reaction, Incubation, Lysis, Derivative Assay, Activity Assay, Generated, Real-time Polymerase Chain Reaction

    EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation

    EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation, Electrophoresis

    DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation, Isolation, Agarose Gel Electrophoresis, Staining

    Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli DNA polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.

    Journal: Journal of Virology

    Article Title: Relationship between 3?-Azido-3?-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

    doi: 10.1128/JVI.77.11.6127-6137.2003

    Figure Lengend Snippet: Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli DNA polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.

    Article Snippet: The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (0.3 U; USB Corp.) and all four dNTPs (100 μM each).

    Techniques: Mutagenesis, Incubation, Radioactivity, Concentration Assay

    Nicking enzyme-mediated labeling of open chromatin. a Nicking of crosslinked chromatin using varying amounts of Nt.CviPII. A 1% agarose gel showing differential nicking of HCT116 genomic DNA based on amount of nicking enzyme (10 U, 5 U, 2.5 U, 1 U, 0.3 U, 0 U). M is a DNA molecular weight ladder. b Open chromatin labeling in fixed HeLa cells using dNTPs supplemented with TexasRed-dATP. Top panel : labeling reaction performed in the presence of Nt.CviPII and DNA polymerase I. Middle panel : labeling reaction performed in the presence of DNA polymerase I only. Bottom panel : labeling reaction performed in the absence of Nt.CviPII and DNA polymerase I. TexasRed-dATP was included in all reactions. DNA staining was performed using DAPI ( blue ) and TexasRed stain ( red ) represents labeled OCSs. Magenta stain ( Merge ) represents the colocalization. c Labeling efficiency of OCSs in all three assayed conditions. The y-axis represents the ratio of the intensity of the red pixels to the intensity of the blue pixels ( OCS labeling efficiency ). d Dot blot showing labeling of open chromatin by Nt.CviPII nicking enzyme in both native and formaldehyde-fixed HCT116 cells. The level of labeling was revealed using HRP-conjugated goat anti-biotin antibody

    Journal: Genome Biology

    Article Title: NicE-seq: high resolution open chromatin profiling

    doi: 10.1186/s13059-017-1247-6

    Figure Lengend Snippet: Nicking enzyme-mediated labeling of open chromatin. a Nicking of crosslinked chromatin using varying amounts of Nt.CviPII. A 1% agarose gel showing differential nicking of HCT116 genomic DNA based on amount of nicking enzyme (10 U, 5 U, 2.5 U, 1 U, 0.3 U, 0 U). M is a DNA molecular weight ladder. b Open chromatin labeling in fixed HeLa cells using dNTPs supplemented with TexasRed-dATP. Top panel : labeling reaction performed in the presence of Nt.CviPII and DNA polymerase I. Middle panel : labeling reaction performed in the presence of DNA polymerase I only. Bottom panel : labeling reaction performed in the absence of Nt.CviPII and DNA polymerase I. TexasRed-dATP was included in all reactions. DNA staining was performed using DAPI ( blue ) and TexasRed stain ( red ) represents labeled OCSs. Magenta stain ( Merge ) represents the colocalization. c Labeling efficiency of OCSs in all three assayed conditions. The y-axis represents the ratio of the intensity of the red pixels to the intensity of the blue pixels ( OCS labeling efficiency ). d Dot blot showing labeling of open chromatin by Nt.CviPII nicking enzyme in both native and formaldehyde-fixed HCT116 cells. The level of labeling was revealed using HRP-conjugated goat anti-biotin antibody

    Article Snippet: Open chromatin DNA was labeled with biotin by incubating the nuclei in the presence of 2.5 U of Nt.CviPII (NEB R0626S), 10 U of DNA polymerase I (M0209S) and 30 μM of each dNTP including 6 μM of biotin-14-dATP (Invitrogen, 19524-016) and 6 μM of biotin-16-dCTP (ChemCyte, CC-6003-1) in 200 μL of 1× NEB buffer 2.

    Techniques: Labeling, Agarose Gel Electrophoresis, Molecular Weight, Staining, Dot Blot

    Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).

    Journal: DNA repair

    Article Title: Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction

    doi: 10.1016/j.dnarep.2016.05.017

    Figure Lengend Snippet: Comparison effects of Mg 2+ concentration on the activity of polα-prim and the Klenow fragment on the template with random sequence Sequence of the 3’ portion of the template 73b (excludes the 5’ primer-binding region) that can potentially form two hairpins (marked by * and **) is shown. The extension of hetero-DNA primers annealed with the template contains a heterogeneous sequence (73b) by polα-prim (enzyme to primer/template ratio = 1:15) and the Klenow fragment (enzyme to primer/template ratio = 1: 5) in the absence or presence of 0.2–16.0 mM Mg 2+ . Reactions were carried out at 35 °C for three minutes (polα-prim) and one minute (Klenow fragment).

    Article Snippet: A large (Klenow) fragment of E. coli DNA Polymerase I (#M0210S; 5,000 units/ml) was purchased from New England Biolabs Inc., Ipswich, Massachusetts.

    Techniques: Concentration Assay, Activity Assay, Sequencing, Binding Assay

    Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).

    Journal: DNA repair

    Article Title: Divalent ions attenuate DNA synthesis by human DNA polymerase α by changing the structure of the template/primer or by perturbing the polymerase reaction

    doi: 10.1016/j.dnarep.2016.05.017

    Figure Lengend Snippet: Comparison of the effects of Zn 2+ alone and in combination with Mg 2+ on DNA synthesis by polα-prim and the Klenow fragment (A) Extension of hetero-DNA primers annealed with heterogeneous 73b template by polα-prim (enzyme to primer/template ratio = 1:10) and (B) the Klenow fragment. (enzyme to primer/template ratio = 1:5). Lanes 2–9, polα-prim for one minute; Lanes 10–17, polα-prim for eight minutes; Lanes 18–25, the Klenow fragment for one minute; all with 100 µM dNTP at 35 °C. Reactions contained no enzyme (lane 1), no additional Me 2+ (lanes 2, 10, 18), 8 mM Mg 2+ (lanes 3, 11, 19), 10 to 50 µM Zn 2+ (lanes 4–6, 12–14, 20–22), and 8 mM Mg 2+ with 10 to 50 µM Zn 2+ (lanes 7–9, 15–17, 23–25).

    Article Snippet: A large (Klenow) fragment of E. coli DNA Polymerase I (#M0210S; 5,000 units/ml) was purchased from New England Biolabs Inc., Ipswich, Massachusetts.

    Techniques: DNA Synthesis

    Neutralization of DNA polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA polymerase I (Klenow fragment).

    Journal: Journal of Virology

    Article Title: Efficient Gap Repair Catalyzed In Vitro by an Intrinsic DNA Polymerase Activity of Human Immunodeficiency Virus Type 1 Integrase

    doi:

    Figure Lengend Snippet: Neutralization of DNA polymerase activity by an anti-integrase MAb. Dilutions of a mouse ascites fluid containing MAb 35 were prepared in phosphate-buffered saline, and 1 μl of the diluted antibody was added to each 25-μl reaction mixture. Half-filled triangles represent reactions with integrase; open triangles represent reactions with E. coli DNA polymerase I (Klenow fragment).

    Article Snippet: CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate} and Escherichia coli DNA polymerase I (Klenow fragment) and the holoenzyme were from Boehringer Mannheim.

    Techniques: Neutralization, Activity Assay

    Loss of E2F1 attenuates DNA repair. A , comet assay expressed as average tail moment on E2F1 +/+ and E2F1 -/- MEFs untreated ( left-hand graph , control), after incubation for 1 h in medium containing 50 μg/ml MMS ( middle graph, 0 h ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 2 h ( right-hand graph, R ). Experiments were performed in duplicate, and measurement of mean tail moment was from 50 cells/slide from 15-20 randomly selected fields representing the whole area of each slide. Statistical analysis was performed using unpaired two-tailed t test on comet tail moments that were determined using Comet Assay II software (Perceptive Instruments; Suffolk, UK). Error bars , S.D. from separate experiments. B , indirect immunofluorescence microscopy on XRCC1 immunostaining ( top ), DNA polymerase I-mediated FITC-dUTP labeling assay ( middle ), and 4′,6-diamidino-2-phenylindole ( DAPI ) staining ( bottom ) performed on E2F1 -/- and E2F1 +/+ MEFs untreated ( C ), after incubation for 1 h in medium containing 50 μg/ml MMS ( 0 h panels ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 6 h ( R panels ). C , percentage of apoptosis as determined by Annexin V staining and flow cytometry 48 h after infection of the indicated cells with adenovirus expressing wild-type E2F1 ( Adeno-E2F1 ) or adenovirus expressing GFP ( Adeno-control ). Error bars , S.D. of triplicate experiments and statistical analysis performed using Student's unpaired two-tailed t test.

    Journal: The Journal of Biological Chemistry

    Article Title: E2F1 Regulates the Base Excision Repair Gene XRCC1 and Promotes DNA Repair *

    doi: 10.1074/jbc.M710296200

    Figure Lengend Snippet: Loss of E2F1 attenuates DNA repair. A , comet assay expressed as average tail moment on E2F1 +/+ and E2F1 -/- MEFs untreated ( left-hand graph , control), after incubation for 1 h in medium containing 50 μg/ml MMS ( middle graph, 0 h ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 2 h ( right-hand graph, R ). Experiments were performed in duplicate, and measurement of mean tail moment was from 50 cells/slide from 15-20 randomly selected fields representing the whole area of each slide. Statistical analysis was performed using unpaired two-tailed t test on comet tail moments that were determined using Comet Assay II software (Perceptive Instruments; Suffolk, UK). Error bars , S.D. from separate experiments. B , indirect immunofluorescence microscopy on XRCC1 immunostaining ( top ), DNA polymerase I-mediated FITC-dUTP labeling assay ( middle ), and 4′,6-diamidino-2-phenylindole ( DAPI ) staining ( bottom ) performed on E2F1 -/- and E2F1 +/+ MEFs untreated ( C ), after incubation for 1 h in medium containing 50 μg/ml MMS ( 0 h panels ), and after incubation for 1 h in medium containing 50 μg/ml MMS followed by recovery with incubation in fresh drug-free medium for 6 h ( R panels ). C , percentage of apoptosis as determined by Annexin V staining and flow cytometry 48 h after infection of the indicated cells with adenovirus expressing wild-type E2F1 ( Adeno-E2F1 ) or adenovirus expressing GFP ( Adeno-control ). Error bars , S.D. of triplicate experiments and statistical analysis performed using Student's unpaired two-tailed t test.

    Article Snippet: Cells were rinsed in phosphate-buffered saline, permeabilized with 1% Triton X-100 for 20 min, and then incubated in a moist air chamber at 37 °C for 90 min in a labeling mixture containing 10 μ m each dGTP, dATP, and dCTP and 7 μ m dTTP, 3 μ m FITC-dUTP, 20 units/ml Escherichia coli DNA polymerase I (Sigma) in reaction buffer containing 5 m m MgCl2 , 10 m m 2-mercaptoethanol, and 20 μg/ml bovine serum albumin.

    Techniques: Single Cell Gel Electrophoresis, Incubation, Two Tailed Test, Software, Immunofluorescence, Microscopy, Immunostaining, Labeling, Staining, Flow Cytometry, Cytometry, Infection, Expressing