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  • 99
    New England Biolabs t4 pnk
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4190 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher klenow dna polymerase i fragment
    Klenow Dna Polymerase I Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dna polymerase i
    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS <t>DNA</t> synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 2910 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher klenow
    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS <t>DNA</t> synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.
    Klenow, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    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: 97/100, based on 121 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Journal: Journal of Virological Methods

    Article Title: HexaPrime: A novel method for detection of coronaviruses

    doi: 10.1016/j.jviromet.2012.11.039

    Figure Lengend Snippet: Evaluation of the HexaPrime assay. (A) Evaluation of different primer pairs for the detection of coronaviruses. Analysis was conducted using the HCoV-NL63 virus and all primer sets given in Table 2 were tested. Only amplification with primer sets 2, 4, 5 and 8 yielded distinct bands. Sequencing of products and analysis of fragment size revealed that only primer set 2 allowed efficient amplification of the desired product. M: size marker; mock-infected (−) or HCoV-NL63-infected (+) cell culture supernatant. (B) Detection of HCoV-NL63 and HCoV-HKU1 with the HexaPrime assay using primer set 2. All experimental procedures were conducted as described in Section 2 . M: size marker; W: water; NL63 and HKU1: mock-infected (−) or virus-infected (+) cell culture supernatant. (C) Sensitivity of the HexaPrime assay. Concentrated samples containing viral RNA (10 9 copies ml −1 ) were subjected to 10-fold serial dilutions in cell culture supernatant and the HexaPrime assay was conducted. For each RNA concentration, three different enzymes for SS DNA synthesis were trialed. A, B and C denote DNA Polymerase I, T7 Polymerase, and Sequenase 2.0, respectively.

    Article Snippet: The efficiency of different SS synthesis enzymes, T7 Polymerase (Thermo Scientific, Vilnius, Lithuania), DNA Polymerase I (Thermo Scientific, Vilnius, Lithuania), and Sequenase 2.0 (Affymetrix, United Kingdom), was evaluated by means of densitometry following bands separation on a 1.5% agarose gel.

    Techniques: Amplification, Sequencing, Marker, Infection, Cell Culture, Concentration Assay, DNA Synthesis

    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

    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

    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