dna polymerase i Search Results


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  • 99
    New England Biolabs klenow fragment
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6354 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher klenow fragment
    Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the <t>Klenow</t> fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 <t>P]dCTP,</t> and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.
    Klenow Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2548 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dna polymerase i klenow fragment
    Sensitive detection of purified <t>DNA</t> polymerase using DPE-PCR. ( A ) A commercial source of DNA <t>polymerase</t> 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, <t>Klenow,</t> 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.
    Dna Polymerase I Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 838 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa klenow fragment
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna polymerase alpha
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Dna Polymerase Alpha, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher klenow dna polymerase i fragment
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Dna Polymerase I Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher klenow fragment polymerase
    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, <t>Klenow</t> fragment and Klenow fragment <t>Exo-</t> were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Klenow Fragment Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Roche klenow fragment dna polymerase i
    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, <t>Klenow</t> fragment and Klenow fragment <t>Exo-</t> were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Klenow Fragment Dna Polymerase I, supplied by Roche, used in various techniques. Bioz Stars score: 80/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dna polymerase
    End protection by <t>DNA</t> ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a <t>non-ligatable</t> 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3856 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    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: 99/100, based on 156 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Promega klenow dna polymerase i fragment
    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.
    Klenow Dna Polymerase I Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 84/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Article Snippet: Both polymerases could replace UL30-UL42, with Klenow Fragment generating products ~1 kB long.

    Techniques: Agarose Gel Electrophoresis

    Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Article Snippet: Both polymerases could replace UL30-UL42, with Klenow Fragment generating products ~1 kB long.

    Techniques: Agarose Gel Electrophoresis

    A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Journal: Human Genomics

    Article Title: Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

    doi: 10.1186/s40246-018-0160-8

    Figure Lengend Snippet: A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Article Snippet: DNA Polymerase I Large (Klenow) Fragment, restriction enzymes and T4 DNA Ligase were obtained from New England Biolabs (NEB), USA. dNTP mix was purchased from Promega, USA.

    Techniques: Ligation

    Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.

    Journal: Journal of Virology

    Article Title: A Heterogeneous Nuclear Ribonucleoprotein A/B-Related Protein Binds to Single-Stranded DNA near the 5? End or within the Genome of Feline Parvovirus and Can Modify Virus Replication

    doi:

    Figure Lengend Snippet: Effect of DBP40 on the in vitro DNA filled-in of FPV ssDNA by the Klenow fragment of DNA polymerase I. Viral DNA recovered from purified virions was incubated with the polymerase in the presence of deoxynucleoside triphosphates, [ 32 P]dCTP, and either 0 or 50 ng of DBP40. (A) The product generated was electrophoresed in a 1% agarose gel, with the number of disintegrations per minute (in phosphorimager units) in the total DNA product shown. (B) dsDNA produced was digested with Sna I (nt 289) or Bse RI (nt 469) and electrophoresed in a 5% nondenaturing acrylamide gel, which was exposed to X-ray film. Incorporation into the lower band is shown below each lane; size markers are indicated in base pairs. (C) Positions of Sna I and Bse RI sites relative to the 3′-end palindrome of the FPV ssDNA genome.

    Article Snippet: Two units of the Klenow fragment (Gibco/BRL), 50 μM each dATP, dGTP, dCTP, and dTTP, and 5 μCi of [α-32 P]dCTP (4 Ci/mmol) were added, and the reaction mixture was incubated for a further 20 min at 37°C; 20 mM EDTA and 1% sodium dodecyl sulfate were added to stop the reaction.

    Techniques: In Vitro, Purification, Incubation, Generated, Agarose Gel Electrophoresis, Produced, Acrylamide Gel Assay

    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

    Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Journal: Scientific Reports

    Article Title: Unusual isothermal multimerization and amplification by the strand-displacing DNA polymerases with reverse transcription activities

    doi: 10.1038/s41598-017-13324-0

    Figure Lengend Snippet: Verification of UIMA using different DNA polymerases. All reactions shared the same primer (RL) and template (F*R*) and were incubated for 180 min. The sequences of RL and F*R* were shown in Table S1 . ( A ) Real-time fluorescence change in reactions using a series of Bst DNA polymerases ( Bst LF, Bst 2.0, Bst 2.0 WS, and Bst 3.0) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( B ) Real-time fluorescence change in reactions using non- Bst polymerases (Bsm, BcaBEST, Vent(exo-), and z-Taq) at 63 °C. No-primer controls (NPCs) were shown in Fig. S5 . ( C ) Temperature gradients assay for the products of reactions using the polymerases with negative results in ( B ). The products were analyzed by 2.5% agarose gel electrophoresis. NTC and NPC for Bsm were performed at 56 °C. NTCs and NPCs for Vent (exo-) and z-Taq were performed at 63 °C. The groping of gels cropped from different gels. Exposure time is 5 s. ( D ) Temperature gradients assay for the products of reactions using the polymerases of Klenow(exo-) and Klenow. The products were analyzed by 2.5% agarose gel electrophoresis. Their NTCs and NPCs were performed at 43 °C. M1 and M2: DNA Marker. NTC: no-target control; NPC: no-primer control. The groping of gels cropped from different gels. Exposure time is 5 s. The full-length gels are presented in Supplementary Figure S7 .

    Article Snippet: General information Bst DNA polymerase Large fragment (Bst LF), Bst 2.0 DNA polymerase (Bst 2.0), Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS), Bst 3.0 DNA polymerase (Bst 3.0), Klenow fragment polymerase (Klenow), Klenow fragment exo- polymerase (Klenow (exo-)), Vent exo- DNA polymerase (Vent (exo-)), and dNTP Mix were purchased from New England Biolabs.

    Techniques: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

    Detection of different concentrations of target based on isothermal strand-displacement polymerization reaction. Experiments were performed in the presence of 15U polymerase Klenow fragment exo − and 100 μM dNTPs with 5 × 10 –8 M probe, 5 × 10 –8 M primer, and different concentrations of target. ( A ) Monitoring the fluorescence intensity of this amplified DNA detection method over a range of target DNA concentrations. The curves from a to i contain the target with 1.0 × 10 –10 , 2.0 × 10 –11 , 4.0 × 10 –12 , 8.0 × 10 –13 , 1.6 × 10 –13 , 3.2 × 10 –14 , 6.4 × 10 –15 , 1.28 × 10 –15 and 0 M, respectively. All samples were incubated at 37°C. ( B ) The relationship of the rate of fluorescence enhancement with target DNA concentration.

    Journal: Nucleic Acids Research

    Article Title: Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction

    doi: 10.1093/nar/gkn1024

    Figure Lengend Snippet: Detection of different concentrations of target based on isothermal strand-displacement polymerization reaction. Experiments were performed in the presence of 15U polymerase Klenow fragment exo − and 100 μM dNTPs with 5 × 10 –8 M probe, 5 × 10 –8 M primer, and different concentrations of target. ( A ) Monitoring the fluorescence intensity of this amplified DNA detection method over a range of target DNA concentrations. The curves from a to i contain the target with 1.0 × 10 –10 , 2.0 × 10 –11 , 4.0 × 10 –12 , 8.0 × 10 –13 , 1.6 × 10 –13 , 3.2 × 10 –14 , 6.4 × 10 –15 , 1.28 × 10 –15 and 0 M, respectively. All samples were incubated at 37°C. ( B ) The relationship of the rate of fluorescence enhancement with target DNA concentration.

    Article Snippet: The polymerase Klenow fragment exo− was purchased from New England Biolabs, Inc.

    Techniques: Fluorescence, Amplification, Incubation, Concentration Assay

    The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Article Snippet: A fragment containing MxA gene was prepared from pET14b-MxA by digestion with NdeI followed by treatment with Klenow fragment and digestion with BamHI.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Journal: PLoS ONE

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    doi: 10.1371/journal.pone.0052584

    Figure Lengend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Article Snippet: Enzymes used These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

    Techniques: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    Left panel: kinetics of Flu emission change during the extension of G-strand of Flu-(dC) 220 –(dG) 220 -TAMRA by Klenow exo − . The reaction was started by the addition of 20 µg/ml Klenow exo − to the cuvette containing 100 mM Tris-Acetate, pH 8.0, 1.2 mM MgCl 2 , 5 mM DTT, 1.0 mM dGTP and 0.2 µM Flu-(dG) 220 –TAMRA-(dC) 220 duplex and followed in time at 37°C by monitoring Flu emission at 520 nm; excitation was at 490 nm. Schematic presentation of the intermediate products of the synthesis is indicated to the right: F denotes for Flu, T for TAMRA. Emission of Flu in 220 bp long poly(dG)–poly(dC) is not quenched by TAMRA attached at the opposite end of the DNA molecule. Extension of the G-strand (new bases incorporated into the polymer are marked in red) results in folding the strand back and, as a result, in decrease of the molecular distance separating the dyes. This phase of the strand extension (phase 1) is not associated with a decrease of Flu emission, since the dyes are still positioned far away from one another and cannot communicate via FRET; further decrease of the separation distance (phase 2) results in FRET between the dyes and in a stepwise drop of the Flu emission. The expansion of the strand is stopped when a complete intramolecular triplex is formed (phase 3). In the triplex, the dyes are positioned close to one another and thus efficiently communicate via FRET.

    Journal: Nucleic Acids Research

    Article Title: Synthesis of novel poly(dG)-poly(dG)-poly(dC) triplex structure by Klenow exo− fragment of DNA polymerase I

    doi: 10.1093/nar/gki963

    Figure Lengend Snippet: Left panel: kinetics of Flu emission change during the extension of G-strand of Flu-(dC) 220 –(dG) 220 -TAMRA by Klenow exo − . The reaction was started by the addition of 20 µg/ml Klenow exo − to the cuvette containing 100 mM Tris-Acetate, pH 8.0, 1.2 mM MgCl 2 , 5 mM DTT, 1.0 mM dGTP and 0.2 µM Flu-(dG) 220 –TAMRA-(dC) 220 duplex and followed in time at 37°C by monitoring Flu emission at 520 nm; excitation was at 490 nm. Schematic presentation of the intermediate products of the synthesis is indicated to the right: F denotes for Flu, T for TAMRA. Emission of Flu in 220 bp long poly(dG)–poly(dC) is not quenched by TAMRA attached at the opposite end of the DNA molecule. Extension of the G-strand (new bases incorporated into the polymer are marked in red) results in folding the strand back and, as a result, in decrease of the molecular distance separating the dyes. This phase of the strand extension (phase 1) is not associated with a decrease of Flu emission, since the dyes are still positioned far away from one another and cannot communicate via FRET; further decrease of the separation distance (phase 2) results in FRET between the dyes and in a stepwise drop of the Flu emission. The expansion of the strand is stopped when a complete intramolecular triplex is formed (phase 3). In the triplex, the dyes are positioned close to one another and thus efficiently communicate via FRET.

    Article Snippet: Klenow fragment exonuclease minus of DNA polymerase I from Escherichia coli lacking the 3′→5′exonuclease activity (Klenow exo− ) was purchased from Fermentas (Lithuania).

    Techniques:

    HPLC analysis of poly(dG–dG)–poly(dC) synthesis. ( A ) Size-dependent HPLC separation of the products of the synthesis. Polymerase extension assay was performed as described in ‘Materials and Methods’, with 2 µM 700 bp poly(dG)–poly(dC), 2.5 mM dGTP, 3.5 mM Mg 2+ and 10 µg/ml Klenow exo − at 37°C. The reaction was started by addition of the enzyme. Aliquots of 50 µl were withdrawn from the assay mixture before (solid curve) and 3 h after (dashed curve) the addition of the enzyme, and loaded on TSKgel G-DNA-PW column (7.8 × 300 mm). Elution was performed with 20 mM Tris-Acetate buffer, pH 7.0, at a flow rate of 0.5 ml/min. ( B ) Time course of dGTP consumption. Polymerase extension assay was performed as described in (A). Aliquots were withdrawn from the assay after every hour and chromatographed as shown in (A). Nucleotide peaks from size-exclusion separations were collected and the amount of dGTP in the peaks was measured by absorption spectroscopy as described in ‘Materials and Methods’. The amount of dGTP in the assay is plotted against time of synthesis. ( C ) Size-dependent HPLC of poly(dG)–poly(dC) and poly(dG–dG)–poly(dC) at high pH. Poly(dG–dG)–poly(dC) was synthesized as described in (A). Initial poly(dG)–poly(dC) (solid curve) and poly(dG–dG)–poly(dC) derived from extension of G-strand in the poly(dG)–Poly(dC) (dashed curve) were pretreated for 15 min at room temperature in 0.1 M KOH. A total of 100 (l of each DNA sample were applied onto TSKgel G-DNA-PW column (7.8 × 300 mm) and eluted with 0.1 M KOH at a flow rate of 0.5 ml/min.

    Journal: Nucleic Acids Research

    Article Title: Synthesis of novel poly(dG)-poly(dG)-poly(dC) triplex structure by Klenow exo− fragment of DNA polymerase I

    doi: 10.1093/nar/gki963

    Figure Lengend Snippet: HPLC analysis of poly(dG–dG)–poly(dC) synthesis. ( A ) Size-dependent HPLC separation of the products of the synthesis. Polymerase extension assay was performed as described in ‘Materials and Methods’, with 2 µM 700 bp poly(dG)–poly(dC), 2.5 mM dGTP, 3.5 mM Mg 2+ and 10 µg/ml Klenow exo − at 37°C. The reaction was started by addition of the enzyme. Aliquots of 50 µl were withdrawn from the assay mixture before (solid curve) and 3 h after (dashed curve) the addition of the enzyme, and loaded on TSKgel G-DNA-PW column (7.8 × 300 mm). Elution was performed with 20 mM Tris-Acetate buffer, pH 7.0, at a flow rate of 0.5 ml/min. ( B ) Time course of dGTP consumption. Polymerase extension assay was performed as described in (A). Aliquots were withdrawn from the assay after every hour and chromatographed as shown in (A). Nucleotide peaks from size-exclusion separations were collected and the amount of dGTP in the peaks was measured by absorption spectroscopy as described in ‘Materials and Methods’. The amount of dGTP in the assay is plotted against time of synthesis. ( C ) Size-dependent HPLC of poly(dG)–poly(dC) and poly(dG–dG)–poly(dC) at high pH. Poly(dG–dG)–poly(dC) was synthesized as described in (A). Initial poly(dG)–poly(dC) (solid curve) and poly(dG–dG)–poly(dC) derived from extension of G-strand in the poly(dG)–Poly(dC) (dashed curve) were pretreated for 15 min at room temperature in 0.1 M KOH. A total of 100 (l of each DNA sample were applied onto TSKgel G-DNA-PW column (7.8 × 300 mm) and eluted with 0.1 M KOH at a flow rate of 0.5 ml/min.

    Article Snippet: Klenow fragment exonuclease minus of DNA polymerase I from Escherichia coli lacking the 3′→5′exonuclease activity (Klenow exo− ) was purchased from Fermentas (Lithuania).

    Techniques: High Performance Liquid Chromatography, Flow Cytometry, Spectroscopy, Synthesized, Derivative Assay

    End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    doi:

    Figure Lengend Snippet: End protection by DNA ligase IV–XRCC4 protein can occur in the absence of DNA end joining. Protein extracts (40 µg) prepared from control lymphoblastoid cell lines, AHH1 and Nalm-6, the LIG4 syndrome cell line LB2304 and the LIG4 -null cell line N114P2 were incubated with a non-ligatable 5′- 32 P-end-labeled substrate (20 ng). Recombinant DNA ligase IV–XRCC4 (180 ng) was added where shown. Product formation was analyzed by agarose gel electrophoresis followed by autoradiography.

    Article Snippet: To generate a non-ligatable substrate, a single nucleotide (dTTP) was incorporated using DNA polymerase I large Klenow fragment (New England Biolabs).

    Techniques: Incubation, Labeling, Recombinant, Agarose Gel Electrophoresis, Autoradiography

    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