klenow fragment Search Results


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  • 99
    New England Biolabs klenow fragment
    Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and <t>Klenow</t> fragment. D) Single digested with Bgl II. E) Random ligation with T4 <t>DNA</t> ligase.
    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 dna polymerase i
    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear <t>DNA</t> using DNA <t>polymerase</t> I and Ampligase. This process substantially improved the complexity of the library during sequencing.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1977 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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 690 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa dna polymerase i klenow
    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 I Klenow, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna polymerase i klenow
    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 I Klenow, 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
    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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    86
    Roche klenow fragment dna polymerase i
    Strategy used to transfer cDNA from Gateway entry vectors to L. lactis vectors. A. Overview of the cloning procedure. In this strategy, recombination occurs between the att L sites from Gateway entry vectors (containing the cDNA coding for the proteins of interest) and the att R sites from a “destination” vector (att: attachment sites). This destination vector (pBS-RfA) is a derivative of pBlueScript which contains the reading frame cassette A (RfA cassette) surrounded by two Eco RV sites. By LR recombination, the first step thus generates a “shuttle” vector in which the gene of interest is surrounded by the two att B sites and two flanking Eco RV restriction sites. In order to generate blunt ends, the nisin inducible vector pNZ8148 is digested by Nco I and treated with the <t>Klenow</t> enzyme <t>(pNZ8148NK).</t> The cDNA excised from the “shuttle” vector pBS-RfA-cDNA with Eco RV (generating blunt ends) is ligated into the vector pNZ8148NK and placed under the control of the P nisA promoter. Positive selection of recombinant vectors is obtained using digestion of the ligation products with NsiI. B. Resulting N - and C - termini of the ORF. Nucleotide sequences at each side of the ORF (upper panel: 5′ORF and lower panel: 3′ORF). In bold, the ATG (start codon) and TAG (stop codon) of the cDNA. CCATG in bold corresponds to the Nco I site after digestion and treatment with the Klenow enzyme; the ATG within the Nco I site is in the reading frame of the ATG of the cDNA. The att B sites are in italics and the two half-sites resulting from Eco RV digestion are in bold italics.
    Klenow Fragment Dna Polymerase I, supplied by Roche, used in various techniques. Bioz Stars score: 86/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega klenow dna polymerase i fragment
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Dna Polymerase I Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 9 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 85 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher klenow
    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, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 471 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t4 polynucleotide kinase
    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.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 24211 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 160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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.
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    Enzymatics dna polymerase i
    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 I, supplied by Enzymatics, used in various techniques. Bioz Stars score: 94/100, based on 105 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc dna polymerase i
    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 I, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 99/100, based on 779 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Toyobo klenow fragment
    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.
    Klenow Fragment, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene klenow fragment
    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.
    Klenow Fragment, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 240 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa e coli dna polymerase i
    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.
    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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    91
    Level Biotechnology Inc klenow fragment
    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.
    Klenow Fragment, supplied by Level Biotechnology Inc, used in various techniques. Bioz Stars score: 91/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GE Healthcare klenow fragment
    Identification of proteins recognizing myc P2 double-stranded, but not single-stranded DNA. A double-stranded probe containing the TFO target and flanking sequence was labeled using [ 32 <t>P]dCTP</t> and the <t>Klenow</t> fragment of DNA polymerase then incubated with nuclear extracts from MCF-7 breast cancer cells. Protein–DNA complexes were resolved on 4% polyacrylamide non-denaturing gels at 10°C in 0.5× TBE. ( A ) Nuclear extracts were pre-incubated with rabbit polyclonal antibodies against Sp1 or Sp3, or with normal rabbit IgG, before adding Myc P2 DNA probe, incubating again then resolving complexes by gel electrophoresis. Arrowheads and arrows indicate Sp1 and Sp3 protein complexes, respectively. ( B ) Single-stranded oligos were added to labeled P2 probe simultaneously with nuclear extracts, incubated, then resolved on a gel. U, unbound DNA probe; ns, band representing protein(s) binding non-specifically to P2 probe.
    Klenow Fragment, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 92/100, based on 410 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MBI Corporation klenow fragment
    Identification of proteins recognizing myc P2 double-stranded, but not single-stranded DNA. A double-stranded probe containing the TFO target and flanking sequence was labeled using [ 32 <t>P]dCTP</t> and the <t>Klenow</t> fragment of DNA polymerase then incubated with nuclear extracts from MCF-7 breast cancer cells. Protein–DNA complexes were resolved on 4% polyacrylamide non-denaturing gels at 10°C in 0.5× TBE. ( A ) Nuclear extracts were pre-incubated with rabbit polyclonal antibodies against Sp1 or Sp3, or with normal rabbit IgG, before adding Myc P2 DNA probe, incubating again then resolving complexes by gel electrophoresis. Arrowheads and arrows indicate Sp1 and Sp3 protein complexes, respectively. ( B ) Single-stranded oligos were added to labeled P2 probe simultaneously with nuclear extracts, incubated, then resolved on a gel. U, unbound DNA probe; ns, band representing protein(s) binding non-specifically to P2 probe.
    Klenow Fragment, supplied by MBI Corporation, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Mannheim klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 95/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 86/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna polymerase i dnase i
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
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    92
    GeneWorks klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
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    Image Search Results


    Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and Klenow fragment. D) Single digested with Bgl II. E) Random ligation with T4 DNA ligase.

    Journal: Microbial Cell Factories

    Article Title: An evolved xylose transporter from Zymomonas mobilis enhances sugar transport in Escherichia coli

    doi: 10.1186/1475-2859-8-66

    Figure Lengend Snippet: Schematic overview of random deletion used in this study . A) Linearization of the plasmid by restriction digestion at Sca I site. B) Random deletion with Exo III. C) Blunt-ended with mung bean nuclease and Klenow fragment. D) Single digested with Bgl II. E) Random ligation with T4 DNA ligase.

    Article Snippet: To facilitate the re-ligation (see below), the purified DNA was blunt-ended with 3.2 U Klenow fragment in 10× NEBuffer 2 (New England Biolabs) and dNTPs (final concentration 33 μM each nucleotide) for 15 min at 25°C.

    Techniques: Plasmid Preparation, Ligation

    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

    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Journal: Nature biotechnology

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

    doi: 10.1038/nbt.2720

    Figure Lengend Snippet: Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Article Snippet: 10 U of DNA Polymerase I (Invitrogen, Carlsbad, CA) was added to the denatured amplicons along with 250 nanograms of unmodified random hexamer primer, 1 mM dNTPs, 1x Ampligase buffer (Epicentre, Madison, Wi), and 1x NEB buffer 2 (NEB, Cambridge, MA).

    Techniques: Amplification, Lysis, Neutralization, Multiple Displacement Amplification, Transferring, Microscopy, Fluorescence, Micromanipulation, Electron Microscopy, Incubation, Evaporation, Sequencing

    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

    Strategy used to transfer cDNA from Gateway entry vectors to L. lactis vectors. A. Overview of the cloning procedure. In this strategy, recombination occurs between the att L sites from Gateway entry vectors (containing the cDNA coding for the proteins of interest) and the att R sites from a “destination” vector (att: attachment sites). This destination vector (pBS-RfA) is a derivative of pBlueScript which contains the reading frame cassette A (RfA cassette) surrounded by two Eco RV sites. By LR recombination, the first step thus generates a “shuttle” vector in which the gene of interest is surrounded by the two att B sites and two flanking Eco RV restriction sites. In order to generate blunt ends, the nisin inducible vector pNZ8148 is digested by Nco I and treated with the Klenow enzyme (pNZ8148NK). The cDNA excised from the “shuttle” vector pBS-RfA-cDNA with Eco RV (generating blunt ends) is ligated into the vector pNZ8148NK and placed under the control of the P nisA promoter. Positive selection of recombinant vectors is obtained using digestion of the ligation products with NsiI. B. Resulting N - and C - termini of the ORF. Nucleotide sequences at each side of the ORF (upper panel: 5′ORF and lower panel: 3′ORF). In bold, the ATG (start codon) and TAG (stop codon) of the cDNA. CCATG in bold corresponds to the Nco I site after digestion and treatment with the Klenow enzyme; the ATG within the Nco I site is in the reading frame of the ATG of the cDNA. The att B sites are in italics and the two half-sites resulting from Eco RV digestion are in bold italics.

    Journal: PLoS ONE

    Article Title: Lactococcus lactis, an Alternative System for Functional Expression of Peripheral and Intrinsic Arabidopsis Membrane Proteins

    doi: 10.1371/journal.pone.0008746

    Figure Lengend Snippet: Strategy used to transfer cDNA from Gateway entry vectors to L. lactis vectors. A. Overview of the cloning procedure. In this strategy, recombination occurs between the att L sites from Gateway entry vectors (containing the cDNA coding for the proteins of interest) and the att R sites from a “destination” vector (att: attachment sites). This destination vector (pBS-RfA) is a derivative of pBlueScript which contains the reading frame cassette A (RfA cassette) surrounded by two Eco RV sites. By LR recombination, the first step thus generates a “shuttle” vector in which the gene of interest is surrounded by the two att B sites and two flanking Eco RV restriction sites. In order to generate blunt ends, the nisin inducible vector pNZ8148 is digested by Nco I and treated with the Klenow enzyme (pNZ8148NK). The cDNA excised from the “shuttle” vector pBS-RfA-cDNA with Eco RV (generating blunt ends) is ligated into the vector pNZ8148NK and placed under the control of the P nisA promoter. Positive selection of recombinant vectors is obtained using digestion of the ligation products with NsiI. B. Resulting N - and C - termini of the ORF. Nucleotide sequences at each side of the ORF (upper panel: 5′ORF and lower panel: 3′ORF). In bold, the ATG (start codon) and TAG (stop codon) of the cDNA. CCATG in bold corresponds to the Nco I site after digestion and treatment with the Klenow enzyme; the ATG within the Nco I site is in the reading frame of the ATG of the cDNA. The att B sites are in italics and the two half-sites resulting from Eco RV digestion are in bold italics.

    Article Snippet: The three independent cDNAs were excised from the pBS-RfA-cDNA vector by digestion with Eco RV and ligated into pNZ8148NK. pNZ8148NK was obtained by digestion with Nco I and subsequent treatment by Klenow fragment (Roche, Switzerland) which filled the cohesive ends.

    Techniques: Clone Assay, Plasmid Preparation, Selection, Recombinant, Ligation

    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

    Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: End Labeling, Labeling

    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

    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

    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

    Identification of proteins recognizing myc P2 double-stranded, but not single-stranded DNA. A double-stranded probe containing the TFO target and flanking sequence was labeled using [ 32 P]dCTP and the Klenow fragment of DNA polymerase then incubated with nuclear extracts from MCF-7 breast cancer cells. Protein–DNA complexes were resolved on 4% polyacrylamide non-denaturing gels at 10°C in 0.5× TBE. ( A ) Nuclear extracts were pre-incubated with rabbit polyclonal antibodies against Sp1 or Sp3, or with normal rabbit IgG, before adding Myc P2 DNA probe, incubating again then resolving complexes by gel electrophoresis. Arrowheads and arrows indicate Sp1 and Sp3 protein complexes, respectively. ( B ) Single-stranded oligos were added to labeled P2 probe simultaneously with nuclear extracts, incubated, then resolved on a gel. U, unbound DNA probe; ns, band representing protein(s) binding non-specifically to P2 probe.

    Journal: Nucleic Acids Research

    Article Title: Design of a novel triple helix-forming oligodeoxyribonucleotide directed to the major promoter of the c-myc gene

    doi:

    Figure Lengend Snippet: Identification of proteins recognizing myc P2 double-stranded, but not single-stranded DNA. A double-stranded probe containing the TFO target and flanking sequence was labeled using [ 32 P]dCTP and the Klenow fragment of DNA polymerase then incubated with nuclear extracts from MCF-7 breast cancer cells. Protein–DNA complexes were resolved on 4% polyacrylamide non-denaturing gels at 10°C in 0.5× TBE. ( A ) Nuclear extracts were pre-incubated with rabbit polyclonal antibodies against Sp1 or Sp3, or with normal rabbit IgG, before adding Myc P2 DNA probe, incubating again then resolving complexes by gel electrophoresis. Arrowheads and arrows indicate Sp1 and Sp3 protein complexes, respectively. ( B ) Single-stranded oligos were added to labeled P2 probe simultaneously with nuclear extracts, incubated, then resolved on a gel. U, unbound DNA probe; ns, band representing protein(s) binding non-specifically to P2 probe.

    Article Snippet: Double-stranded 32 P-labeled probes with myc P1 and P2 promoter sequences were prepared by annealing complementary, gel-purified single-stranded oligonucleotides then filling in 5′ overhangs using Klenow fragment and [32 P]dCTP (3000 Ci/mmol; Amersham Pharmacia, Piscataway, NJ).

    Techniques: Sequencing, Labeling, Incubation, Nucleic Acid Electrophoresis, Binding Assay

    The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Journal: Nucleic Acids Research

    Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

    doi:

    Figure Lengend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Article Snippet: Exonuclease III (exo III) and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation, Labeling