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  • 99
    New England Biolabs klenow dna polymerase i
    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of <t>DNA</t> <t>polymerase</t> I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Klenow Dna Polymerase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher klenow dna polymerase i fragment
    PAGE analysis of primer extension experiments with single OXP-modified and PDE primers. Primer extension with Klenow fragment of <t>DNA</t> <t>polymerase</t> I of nonheated ( A ) and preheated ( B ) single OXP-modified reverse primer, respectively along template 2. The extension reactions were incubated at 25°C for the indicated times after which the reaction mixtures were quenched and analyzed. ( C ) Primer extension with Taq DNA polymerase of PDE and OXP forward primers (nonheated control and preheated sample) along template oligonucleotide 1. Extension reactions were incubated at 25°C for 15 min, after which the aliquots from reaction mixtures were quenched and analyzed.
    Klenow Dna Polymerase I Fragment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs dna polymerase i klenow fragment
    Sensitive detection of purified <t>DNA</t> polymerase using DPE-PCR. ( A ) A commercial source of DNA <t>polymerase</t> I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, <t>Klenow,</t> Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.
    Dna Polymerase I Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 956 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 2905 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa klenow fragment
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Fragment, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 924 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    TaKaRa klenow fragment dna polymerase i
    The effect of transiently expressed nuclear <t>MxA</t> proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with <t>Klenow</t> fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).
    Klenow Fragment Dna Polymerase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    New England Biolabs large klenow fragment
    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target <t>DNA</t> at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large <t>Klenow</t> fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.
    Large Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 1220 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs klenow dna pol i fragment
    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the <t>Klenow</t> fragment <t>exo</t> (−).
    Klenow Dna Pol I Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 32 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche klenow fragment dna polymerase i
    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the <t>Klenow</t> fragment <t>exo</t> (−).
    Klenow Fragment Dna Polymerase I, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Promega klenow dna polymerase i fragment
    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the <t>Klenow</t> fragment <t>exo</t> (−).
    Klenow Dna Polymerase I Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 88/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher klenow
    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the <t>Klenow</t> fragment <t>exo</t> (−).
    Klenow, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 499 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


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

    Journal: Nucleic Acids Research

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

    doi: 10.1093/nar/gkn575

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

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

    Techniques: Polyacrylamide Gel Electrophoresis, Modification, Incubation

    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

    Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Journal:

    Article Title: Effect of Cross-Link Structure on DNA Interstrand Cross-Link Repair Synthesis

    doi: 10.1021/tx9000896

    Figure Lengend Snippet: Primer extension assay with E. coli Pol I and T7 DNA polymerase. (A) Schematic of the substrates used in the assay. Each template strand contains a site-specific single base cross-link remnant. Star = 32 P label (B) Sequences of the primer and template

    Article Snippet: Protected deoxyribonucleoside-3′-O-methylphosphonamidites were a product of JBL, Inc. Polynucleotide kinase, T4 DNA ligase, E. coli DNA polymerase I (Klenow fragment), and T7 DNA polymerase were obtained from New England Biolabs, Inc. Shrimp alkaline phosphatase (SAP) was from Roche Diagnostics.

    Techniques: Primer Extension Assay

    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

    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

    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Selection, Marker, In Vitro, Transformation Assay, Sequencing, Ligation

    Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Generated, In Vitro, Sequencing, Variant Assay, Ligation

    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Journal: Nucleic Acids Research

    Article Title: Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

    doi: 10.1093/nar/gky452

    Figure Lengend Snippet: Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Article Snippet: The reaction was supplemented with 1 μl (20 units) of Exonuclease I (New England BioLabs) and incubated at 37°C for 15 min. Then, the reaction was supplemented with 1 μl (50 units) of Klenow fragment exo (−) (New England BioLabs) and further incubated for 15 min at 37°C.

    Techniques: Primer Extension Assay