dna polymerase Search Results


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  • 99
    Qiagen dna polymerase
    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start <t>Taq</t> <t>DNA</t> polymerase at same dilutions; lane M 100 bp marker
    Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 505 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs onetaq dna polymerase
    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start <t>Taq</t> <t>DNA</t> polymerase at same dilutions; lane M 100 bp marker
    Onetaq Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 658 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dna polymerase dnapol
    Beta distributions representing enzyme error rates. The model derived beta distributions representing the error rates for the <t>DNA</t> polymerase (blue line) and Reverse Transcriptase (red line) enzymes. 100,000 samples were randomly drawn from each Beta distribution. All error rates are logged (base 10) and the x-axis is truncated at −11 (the RT distribution has a long low density tail stretching to −30) to aid viewing and focus on the differences between the two distributions.
    Dna Polymerase Dnapol, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega gotaq dna polymerase
    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different <t>DNA</t> polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using <t>GoTaq</t> DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Gotaq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 9122 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega taq dnapol
    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different <t>DNA</t> polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using <t>GoTaq</t> DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Taq Dnapol, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Stratagene herculase dna polymerase
    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different <t>DNA</t> polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using <t>GoTaq</t> DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Herculase Dna Polymerase, supplied by Stratagene, used in various techniques. Bioz Stars score: 87/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Millipore taq dnapol
    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different <t>DNA</t> polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using <t>GoTaq</t> DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.
    Taq Dnapol, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher accuprime pfx dna polymerase
    Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and <t>AccuPrime</t> Pfx (Life Technologies) <t>DNA</t> polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .
    Accuprime Pfx Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2231 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa dna polymerase
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1819 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Millipore polymerase
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 111 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Promega pfu dnapol
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Pfu Dnapol, supplied by Promega, used in various techniques. Bioz Stars score: 87/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna polymerase
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 286 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher phusion dna polymerase
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Phusion Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8083 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Agilent technologies pfuultra high fidelity dna polymerase
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Pfuultra High Fidelity Dna Polymerase, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 99/100, based on 619 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Beckman Coulter podo dnapol
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Podo Dnapol, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Zymo Research dna polymerase
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Dna Polymerase, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher amplitaq dna polymerase
    Confronting two-pair primers–polymerase chain reaction for NRF2 -617C/A and -653A/G was carried out at the indicated T a using 2.5U/sample of <t>AmpliTaq</t> Gold polymerase. N: Negative control indicates <t>DNA</t> template omission. MW: Molecular weights (100 bp up to 1000 bp).
    Amplitaq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4433 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher bsm dna polymerase
    Verification of UIMA using different <t>DNA</t> 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 <t>(Bsm,</t> 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 .
    Bsm Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa e2tak dna polymerase
    Verification of UIMA using different <t>DNA</t> 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 <t>(Bsm,</t> 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 .
    E2tak Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 72 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen hotstart dna polymerase
    Impact evaluation of Master Mix used in singleplex PCR. In graph: Elizyme = 2X EliZyme HS Robust MIX (Elisabeth Pharmacon, Czech Republic); Accustart II = AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA); Amplitaq = AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA); OneTaq = OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA); Platinum = Platinum Hot Start PCR 2X Master Mix (Invitrogen, California, USA); <t>HotStarTaq</t> = HotStarTaq <t>DNA</t> Polymerase (Qiagen, Germany). YE = Y . enterocolitica; TG = T . gondii ; plus sign in legend = positive sample; minus sign = NTC.
    Hotstart Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1016 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bio-Rad itaq dna polymerase
    Impact evaluation of Master Mix used in singleplex PCR. In graph: Elizyme = 2X EliZyme HS Robust MIX (Elisabeth Pharmacon, Czech Republic); Accustart II = AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA); Amplitaq = AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA); OneTaq = OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA); Platinum = Platinum Hot Start PCR 2X Master Mix (Invitrogen, California, USA); <t>HotStarTaq</t> = HotStarTaq <t>DNA</t> Polymerase (Qiagen, Germany). YE = Y . enterocolitica; TG = T . gondii ; plus sign in legend = positive sample; minus sign = NTC.
    Itaq Dna Polymerase, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 93/100, based on 781 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs phi29 dna polymerase
    Plasmid <t>DNA</t> from the cecal sample after amplification with <t>phi29</t> polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Phi29 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 607 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    Millipore redtaq dna polymerase
    Plasmid <t>DNA</t> from the cecal sample after amplification with <t>phi29</t> polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Redtaq Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 87/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa seqamp dna polymerase
    Plasmid <t>DNA</t> from the cecal sample after amplification with <t>phi29</t> polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.
    Seqamp Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 dna polymerase
    Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by <t>T4</t> DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 6753 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa t4 dna polymerase
    The PCR product of a foreign gene was amplified by <t>T4</t> DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.
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    New England Biolabs therminator dna polymerase
    TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a <t>DNA</t> template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, <t>Therminator,</t> 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.
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    Qiagen toptaq dna polymerase
    TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a <t>DNA</t> template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, <t>Therminator,</t> 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.
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    Image Search Results


    Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker

    Journal: Indian Journal of Microbiology

    Article Title: Simultaneous Detection of Major Pome Fruit Viruses and a Viroid

    doi: 10.1007/s12088-013-0431-y

    Figure Lengend Snippet: Sensitivity limits of multiplex RT-PCR. Lane 1 Amplification from tenfold diluted cDNA; lanes 2–6 amplicons from 10 −2 to 10 −6 fold diluted cDNA. Lanes 1–6 amplicons obtained with multiplexing kit; lanes 7–12 amplification with hot-start Taq DNA polymerase at same dilutions; lane M 100 bp marker

    Article Snippet: The multiplex RT-PCR assay was optimized with regular Taq (Bangalore Genei, Bengaluru, India), Hot Start Taq (Fermentas, Vilnus, Lithuania) DNA polymerase and multiplexing kit from Qiagen under the conditions described above with nad5 as an internal control.

    Techniques: Multiplex Assay, Reverse Transcription Polymerase Chain Reaction, Amplification, Multiplexing, Marker

    Beta distributions representing enzyme error rates. The model derived beta distributions representing the error rates for the DNA polymerase (blue line) and Reverse Transcriptase (red line) enzymes. 100,000 samples were randomly drawn from each Beta distribution. All error rates are logged (base 10) and the x-axis is truncated at −11 (the RT distribution has a long low density tail stretching to −30) to aid viewing and focus on the differences between the two distributions.

    Journal: BMC Genomics

    Article Title: Distinguishing low frequency mutations from RT-PCR and sequence errors in viral deep sequencing data

    doi: 10.1186/s12864-015-1456-x

    Figure Lengend Snippet: Beta distributions representing enzyme error rates. The model derived beta distributions representing the error rates for the DNA polymerase (blue line) and Reverse Transcriptase (red line) enzymes. 100,000 samples were randomly drawn from each Beta distribution. All error rates are logged (base 10) and the x-axis is truncated at −11 (the RT distribution has a long low density tail stretching to −30) to aid viewing and focus on the differences between the two distributions.

    Article Snippet: The DNA polymerase (DNApol) used in this PCR process was Platinum Taq High Fidelity (Invitrogen).

    Techniques: Derivative Assay

    Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Journal: BMC Biotechnology

    Article Title: FastCloning: a highly simplified, purification-free, sequence- and ligation-independent PCR cloning method

    doi: 10.1186/1472-6750-11-92

    Figure Lengend Snippet: Optimization of cloning conditions . (A) PCR amplification of a target cDNA (human nAChR α9 subunit) and the pGEMHE vector using different DNA polymerases: Pfu Turbo, PfuUltra and Phusion. (B) Comparison of number of colonies grown on the plates after transformation. Three different vector-to-insert ratios (1:1, 1:2, and 1:4) during Dpn I digestion and three amounts of vector-insert mixtures (2, 4, and 8 μl) for transformation were tested. See text for details. (C) Clone validation by PCR using GoTaq DNA polymerase. Lanes 1 to 12: target clones to be validated; Lane 13: 1 Kb plus DNA ladder; Lane 14: pGEMHE vector control; Lane 15: negative control using pCR4-TOPO-α9 parent plasmid. (D) Clone validation by restriction digestion to exclude unusual constructs. Lane 1: 1 Kb plus DNA ladder, Lanes 2-11: target clones double digested with Kpn I and Nhe I. Note that this digestion resulted in a pGEM vector and an insert with α9 nAChR plus the 5'UTR and 3'UTR of Xenopus β-globin.

    Article Snippet: The PCR confirmation of the insert in the target vector (Figure ) was performed with the GoTaq DNA polymerase (Promega Corporation, Madison, WI) using the pGEMHE vector specific primer pair.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Transformation Assay, Negative Control, Construct

    Determination of 3′-phosphatase (A), 5′-phosphatase (B), and 3′-exonuclease (C) activities for YqfS. (A) The ability of either E. coli Nfo (1 U), His 6 -YqfS (YqfS) (500 ng), or alkaline phosphatase (A.P.) (1 U) to stimulate the nick translation activity of DNA containing 3′-phosphate termini was determined as described in Materials and Methods. Error bars show standard deviation. (B) 5′-Phosphatase activity was determined as described in Materials and Methods to either E. coli Nfo (1 U), His 6 -YqfS (500 ng) or alkaline phosphatase (1 U). (C) 3′-Exonuclease activity was determined as described in Materials and Methods to either, E. coli Nfo (1 U), His 6 -YqfS (500 ng), or ExoIII (100 U). C, no added enzyme. For all three panels, the y axis shows counts per minute incorporated (A, 10 5 ; B, 10 5 ; C, 10 4 ). The data are expressed as averages of two independent duplicate determinations.

    Journal: Journal of Bacteriology

    Article Title: YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

    doi: 10.1128/JB.185.18.5380-5390.2003

    Figure Lengend Snippet: Determination of 3′-phosphatase (A), 5′-phosphatase (B), and 3′-exonuclease (C) activities for YqfS. (A) The ability of either E. coli Nfo (1 U), His 6 -YqfS (YqfS) (500 ng), or alkaline phosphatase (A.P.) (1 U) to stimulate the nick translation activity of DNA containing 3′-phosphate termini was determined as described in Materials and Methods. Error bars show standard deviation. (B) 5′-Phosphatase activity was determined as described in Materials and Methods to either E. coli Nfo (1 U), His 6 -YqfS (500 ng) or alkaline phosphatase (1 U). (C) 3′-Exonuclease activity was determined as described in Materials and Methods to either, E. coli Nfo (1 U), His 6 -YqfS (500 ng), or ExoIII (100 U). C, no added enzyme. For all three panels, the y axis shows counts per minute incorporated (A, 10 5 ; B, 10 5 ; C, 10 4 ). The data are expressed as averages of two independent duplicate determinations.

    Article Snippet: Evaluation of other enzymatic properties revealed that YqfS was capable of stimulating the nick translation activity of DNA polymerase by catalyzing the cleavage of 3′ blocking phosphates of DNA treated with micrococcal nuclease.

    Techniques: Nick Translation, Activity Assay, Standard Deviation

    Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Journal: Scientific Reports

    Article Title: PCR amplification of repetitive DNA: a limitation to genome editing technologies and many other applications

    doi: 10.1038/srep05052

    Figure Lengend Snippet: Single strand binding protein, ET SSB, only has a minor effect on the reduction of artifacts. Taq (NE Biolabs) and AccuPrime Pfx (Life Technologies) DNA polymerases were used in amplification of TALE DNA repeats. The arrows indicate the expected size of the amplification products. PCR conditions are given in the supplementary material .

    Article Snippet: Better performance of AccuPrime Pfx DNA polymerase compared to other DNA polymerases on repetitive DNA templates is also likely due to its mixture of other proprietary thermostable accessory proteins in this polymerase mix ( http://tools.lifetechnologies.com/content/sfs/manuals/accuprimepfx_man.pdf ) .

    Techniques: Binding Assay, Amplification, Polymerase Chain Reaction

    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

    doi: 10.1016/j.matbio.2016.04.002

    Figure Lengend Snippet: Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Article Snippet: To identify the mutation in the RCS-Cas9 clone #7, the same PCR product was also amplified but with a different DNA polymerase, TaKaRa LA Taq® DNA polymerase (Clontech), and this PCR product cloned into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Labeling, Clone Assay, Genomic Sequencing

    PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

    Journal: Applied and Environmental Microbiology

    Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

    doi: 10.1128/AEM.69.1.679-682.2003

    Figure Lengend Snippet: PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

    Article Snippet: To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma).

    Techniques: Agarose Gel Electrophoresis, Marker

    a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )

    Journal: Extremophiles

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    doi: 10.1007/s00792-014-0646-9

    Figure Lengend Snippet: a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )

    Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Techniques: Purification, SDS Page, Staining, Incubation, Activity Assay

    Aphidicolin inhibits polB but not polD synthesis. To test if aphidicolin inhibits polB and polD, DNA synthesis was measured as described in “ Materials and Methods ”. a DNA synthesis by polB (10 nM) ( filled squares ) and polD (10 nM) ( filled circles ) was measured in the presence of increasing concentrations of aphidicolin (0–400 μM) and plotted as a percentage of activity in a reaction without aphidicolin. b , c DNA synthesis was also measured over a time course. After 4 min of incubation, aphidicolin (final concentration of 200 μM; open shape ) or dH 2 O ( filled shape ) was added and the reaction was allowed to proceed

    Journal: Extremophiles

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    doi: 10.1007/s00792-014-0646-9

    Figure Lengend Snippet: Aphidicolin inhibits polB but not polD synthesis. To test if aphidicolin inhibits polB and polD, DNA synthesis was measured as described in “ Materials and Methods ”. a DNA synthesis by polB (10 nM) ( filled squares ) and polD (10 nM) ( filled circles ) was measured in the presence of increasing concentrations of aphidicolin (0–400 μM) and plotted as a percentage of activity in a reaction without aphidicolin. b , c DNA synthesis was also measured over a time course. After 4 min of incubation, aphidicolin (final concentration of 200 μM; open shape ) or dH 2 O ( filled shape ) was added and the reaction was allowed to proceed

    Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Techniques: DNA Synthesis, Activity Assay, Incubation, Concentration Assay

    Comparison of error rates and single nucleotide substitutions. a PolD (95 × 10 −5 ) and polD exo- (125 × 10 −5 ) error rates are higher than polB (20 × 10 −5 ). b Transitions ( gray ) are the majority of single base substitutions during synthesis by polD, polD exo- and Taq DNA polymerases. PolD and polD exo- synthesis also yields transversions ( white ) and frameshift deletions ( black ) at lower frequencies

    Journal: Extremophiles

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    doi: 10.1007/s00792-014-0646-9

    Figure Lengend Snippet: Comparison of error rates and single nucleotide substitutions. a PolD (95 × 10 −5 ) and polD exo- (125 × 10 −5 ) error rates are higher than polB (20 × 10 −5 ). b Transitions ( gray ) are the majority of single base substitutions during synthesis by polD, polD exo- and Taq DNA polymerases. PolD and polD exo- synthesis also yields transversions ( white ) and frameshift deletions ( black ) at lower frequencies

    Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Techniques:

    Characterization of polD 3′–5′ exonuclease activity. a FAM-labeled primer/template DNA was incubated in 1× ThermoPol buffer. Various concentrations (10, 5, 2.5, 1.25 nM) of polD wild type ( Lanes 2–5 ), polD/D507A ( Lanes 6–9 ), polD/H554A ( Lanes 10–13 ) or polD/D507A/H554A ( Lanes 14–17 ) were added. Reactions were incubated at 65 °C for 10 min. Reactions omitting polD were run in parallel ( Lane 1 ). Reactions were separated by 15 % TBE-UREA polyacrylamide gel electrophoresis and visualized using a phosphorimager. b DNA polymerase (1 nM) was incubated with a FAM-labeled primer-template (15 nM) in 1× ThermoPol buffer. Exonuclease product was quantified over time and fit to a linear slope to derive rates as described in “ Materials and Methods ”. PolB 3′–5′ exonuclease activity ( filled circle ; 21 fmol/min) was almost two-fold higher than polD ( open circle ; 12 fmol/min). PolD exonuclease-deficient mutants (polD/D507, open square ; polD/H554A, open triangle ; and polD/D507A/H554A, cross hatch ) lacked detectible 3′–5′ exonuclease activity

    Journal: Extremophiles

    Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

    doi: 10.1007/s00792-014-0646-9

    Figure Lengend Snippet: Characterization of polD 3′–5′ exonuclease activity. a FAM-labeled primer/template DNA was incubated in 1× ThermoPol buffer. Various concentrations (10, 5, 2.5, 1.25 nM) of polD wild type ( Lanes 2–5 ), polD/D507A ( Lanes 6–9 ), polD/H554A ( Lanes 10–13 ) or polD/D507A/H554A ( Lanes 14–17 ) were added. Reactions were incubated at 65 °C for 10 min. Reactions omitting polD were run in parallel ( Lane 1 ). Reactions were separated by 15 % TBE-UREA polyacrylamide gel electrophoresis and visualized using a phosphorimager. b DNA polymerase (1 nM) was incubated with a FAM-labeled primer-template (15 nM) in 1× ThermoPol buffer. Exonuclease product was quantified over time and fit to a linear slope to derive rates as described in “ Materials and Methods ”. PolB 3′–5′ exonuclease activity ( filled circle ; 21 fmol/min) was almost two-fold higher than polD ( open circle ; 12 fmol/min). PolD exonuclease-deficient mutants (polD/D507, open square ; polD/H554A, open triangle ; and polD/D507A/H554A, cross hatch ) lacked detectible 3′–5′ exonuclease activity

    Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

    Techniques: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: A safe, programmable system to test CRISPR-based gene editing in haploid yeast. (A) Our design for a yeast system for analysis of CRISPR editing includes (i) an inducible S. pyogenes Cas9 expressed from a URA3 -based plasmid, (ii) a sgRNA expression cassette on a high-copy LEU2 -based plasmid, and (iii) a programmable gene “target” (consisting of a drug resistance marker cassette) at a safe-harbor locus ( HIS3 ) flanked by two “unique” DNA sequences (u1) that do not exist within the S. cerevisiae ). Induction of Cas9 allows targeting and double-stranded break formation at the identical (u1) sequences. In the absence of exogenous DNA ( e.g. ) to the flanking HIS3 UTR. The expected product sizes of the amplified PCR fragments are ∼379 bp (depending on the type of insertion/deletion(s) at the cut site, if any), or 1839 bp in the absence of any editing. Colonies were tested for resistance on medium containing G418 (below). (D) Clonal isolates from Cas9 editing (a dozen independent experiments) using the high copy sgRNA(u1) plasmid from (B) and that had also excised the selection cassette were analyzed by DNA sequencing at the HIS3 locus. The number of each genotype obtained is listed (right).

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: CRISPR, Plasmid Preparation, Expressing, Marker, Amplification, Polymerase Chain Reaction, Selection, DNA Sequencing

    Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 .

    Journal: G3: Genes|Genomes|Genetics

    Article Title: Tuning CRISPR-Cas9 Gene Drives in Saccharomyces cerevisiae

    doi: 10.1534/g3.117.300557

    Figure Lengend Snippet: Altering levels of Cas9 to activate an artificial gene drive in diploid yeast cells. (A) Our design of a programmable gene drive included (i) an integrated copy of S. pyogenes Cas9 (asterisk denotes use of various Cas9 fusions in an otherwise identical construct) under the inducible GAL1/10 promoter at the HIS3 locus in MAT ), and (iv) an artificial gene “target” containing a different selectable marker ( S. pombe HIS5 ) and flanked by (u1) artificial Cas9 sites at the HIS3 locus in a strain of the opposite mating type ( MAT α). (B) Activation and testing of all gene drives was performed as follows. First, the Cas9-containing strain (shown, GFY-2383) was transformed with the sgRNA(u1) plasmid (pGF-IVL1220) or an empty vector (pRS425) control and maintained on dextrose. Second, the gene drive strain ( MAT a) harboring the sgRNA(u1) plasmid was mated to the target strain ( MAT α; GFY-3206 or GFY-3207) on rich medium for 24 hr at 30°. Third, diploid yeast were selected twice on SD-LEU-HIS medium (24 hr incubation at 30°). Fourth, diploids were cultured overnight in S-LEU + Raffinose/Sucrose liquid medium. Fifth, strains were back-diluted to an OD 600 of ∼0.35 OD/ml in YP + Galactose and grown at 30° for various amounts of time. Sixth, yeast were harvested by a brief centrifugation, washed with water, diluted to ∼1000 cells/ml, and 0.5 ml was plated onto SD-LEU medium and incubated at 30° for 2 d. Finally, yeast were transferred by replica-plating to SD-LEU and SD-HIS plates and incubated for 24 additional hours before imaging. Representative plates are shown for the GFY-3206 cross. (C) Quantification of the percentage of colonies displaying an active gene drive (assayed by sensitivity on SD-HIS medium). Error, SD. Statistically significant comparisons are denoted using an unpaired t -test. N.S., not significant. The value for 0 hr is 0% drive activity, not 50%. Experimental runs with an empty plasmid (pRS425) were also performed and displayed a value of zero drive activity for all time points. (D) Clonal isolates were randomly selected from SD-LEU plates from (B) and retested on G418 and SD-HIS media. Multiple crosses were used to determine ploidy status. Diagnostic PCRs (A–D) were performed on isolated diploid chromosomal DNA to assess the HIS3 .

    Article Snippet: Enzymatically dead Cas9 (D10A H840A) was generated by a modified PCR mutagenesis protocol ( ) on the pUC57-based plasmid(s) harboring the Cas9 gene using a high-fidelity DNA polymerase (KOD Hot Start; EMD Millipore).

    Techniques: Construct, Marker, Activation Assay, Transformation Assay, Plasmid Preparation, Incubation, Cell Culture, Centrifugation, Imaging, Activity Assay, Diagnostic Assay, Isolation

    Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

    Journal: Scientific Reports

    Article Title: Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

    doi: 10.1038/s41598-018-19930-w

    Figure Lengend Snippet: Radiation induced mitochondrial biogenesis augments metabolic viability: ( A ) Cell cycle histogram showing phase distribution (G1, S and G2/M) of cells at 24 and 48 hrs post irradiation in HeLa and MDA-MB-231 cells. ( B ) Mitochondrial genome encoded Leu tRNA gene was analyzed by semi quantitative PCR and normalized with nuclear pol gamma gene copy number. The mtDNA copy number is also presented as comparative fold change at respective time points (bar diagram). ( C ) Protein expression analysis of mitochondrial biogenesis and mitochondrial complex-II subunit SDH-A presented in HeLa and MDA-MB-231 cells. The values between the blots represent the fold increase at 8 and 24 hrs. post irradiation quantified by densitometry and normalized with respective β-Actin. The DNA ( B ) and protein blot ( C ). ( D ) Analysis of effect of chloramphenicol (40 μM; 30 mins prior to IR; continuous exposure) on mitochondrial content by MitoTracker Green FM at indicated time points using flow cytometer and graphs presented as fold change of mean fluorescence intensity (MFI) with respective control. Effect of Chloramphenicol on radiation induced growth inhibition was analyzed (at 5 Gy) by MTT assay ( E ) and cell number ( F ) in HeLa and MDA-MB-231 cells. Growth inhibition quantified and mentioned at 48 hours. Data are expressed as mean ± SD from triplicates. *p

    Article Snippet: Primers were purchased from GCC biotech (India) while PCR master mix and hot start DNA polymerase was procured from Sigma, USA.

    Techniques: Irradiation, Multiple Displacement Amplification, Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Cytometry, Fluorescence, Inhibition, MTT Assay

    AR interacts with enzymes of DNA synthesis: A) AR-IP contains DNA polymerase-α. AR-IP prepared from exponentially growing LNCaP cells by using anti-AR mouse monoclonal (441) or rabbit polyclonal (N-20) antibodies was subjected to Western blot analysis. B) AR is colocalized with DNA polymerase-α in LNCaP cells. Exponentially growing LNCaP cells on slides were fixed and stained with anti-AR (N-20) rabbit polyclonal and anti-DNA polymerase-α (STK1) mouse monoclonal antibodies and confocal microscopy was performed. C) AR-IP contains PCNA and ribonucleotide reductase. AR-IP prepared from exponentially growing LNCaP cells was subjected to Western blot analysis. RNR2, ribonucleotide reductase catalytic subunit.

    Journal: PLoS ONE

    Article Title: Role of Androgen Receptor in Progression of LNCaP Prostate Cancer Cells from G1 to S Phase

    doi: 10.1371/journal.pone.0056692

    Figure Lengend Snippet: AR interacts with enzymes of DNA synthesis: A) AR-IP contains DNA polymerase-α. AR-IP prepared from exponentially growing LNCaP cells by using anti-AR mouse monoclonal (441) or rabbit polyclonal (N-20) antibodies was subjected to Western blot analysis. B) AR is colocalized with DNA polymerase-α in LNCaP cells. Exponentially growing LNCaP cells on slides were fixed and stained with anti-AR (N-20) rabbit polyclonal and anti-DNA polymerase-α (STK1) mouse monoclonal antibodies and confocal microscopy was performed. C) AR-IP contains PCNA and ribonucleotide reductase. AR-IP prepared from exponentially growing LNCaP cells was subjected to Western blot analysis. RNR2, ribonucleotide reductase catalytic subunit.

    Article Snippet: Slides were then incubated for 1 hour at 22°C with antibodies against AR (AR-N20 or AR-441) and Cdc6 or DNA polymerase-α followed by both goat-anti-mouse-fluorescein isothiocyanate (FITC)- and goat-anti-rabbit-tetramethylrhodamine B isothiocyanate (TRITC)-labeled secondary antibodies (Sigma-Aldrich, St. Louis, MO).

    Techniques: DNA Synthesis, Western Blot, Staining, Confocal Microscopy

    Screening various DNA polymerases with compound 1A and T1 . Lane 1, primer only; lane 2, ddTTP (control); lane 3, dTTP (control); lane 4, Tth; lane 5, Tfl; lane 6, Thermosequenase; lane 7, Vent(exo-); lane 8, Therminator; lane 9, KOD DASH; lane 10, Klenow; lane 11, Sequenase v.2.0.

    Journal: Molecules

    Article Title: Synthesis and Enzymatic Incorporation of Modified Deoxyuridine Triphosphates

    doi: 10.3390/molecules200813591

    Figure Lengend Snippet: Screening various DNA polymerases with compound 1A and T1 . Lane 1, primer only; lane 2, ddTTP (control); lane 3, dTTP (control); lane 4, Tth; lane 5, Tfl; lane 6, Thermosequenase; lane 7, Vent(exo-); lane 8, Therminator; lane 9, KOD DASH; lane 10, Klenow; lane 11, Sequenase v.2.0.

    Article Snippet: KOD-DASH DNA polymerase was purchased from EMD Millipore (Billerica, MA, USA).

    Techniques:

    Submitochondrial localization of the UCP3/Trx2 interaction. (A) Mitochondria isolated from HeLa cells transfected with UCP3 and Trx2 were suspended in RIPA buffer+SDS or lysis buffer+Triton X-100, respectively. Mitochondrial pellets and supernatants were subjected to SDS-PAGE followed by immunoblotting (IB) with anti-myc, anti-V5, anti-COX4, and anti-DNA polymerase γ (DPγ) antibodies. PPT, mitochondrial pellet after centrifugation; SDS, RIPA buffer; SUP, supernatant after centrifugation; Triton X-100; lysis buffer. (B) . Mitochondrial pellets and supernatants were resuspended in SDS-PAGE buffer, and then loaded onto an SDS-PAGE gel. Immunoblots show the presence of the intermembrane space (IMS) resident Smac and the matrix resident HSP60 in mitochondria that were untreated or treated with proteinase K (Pro K) (lanes 2–7) and increasing concentrations of digitonin (DIG) (lanes 3–7, 0.1 to 0.7 mg/ml DIG). (C) N (VN)- and C (VC)- terminal fragments of Venus fluorescent proteins were fused to the C-terminus of UCP3 (resides 1–308) (UCP3-VN) or ΔCUCP3 (residues1–234) (ΔCUCP3-VN), and the C-terminus of Trx2, respectively. The fragments of fluorescent proteins of belonging to UCP3-VN and ΔCUCP3-VN were localized to the mitochondrial IMS and matrix, respectively. (D) HeLa cells were transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC. Cell extracts (100 μg) were immunoprecipitated (IP) with anti-myc antibodies and analyzed by IB to detect V5 (UCP3) and myc (Trx2). β-Actin was used as an internal standard. (E) Fluorescent images of HeLa cells transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC as indicated in each panel. (F) ).

    Journal: Antioxidants & Redox Signaling

    Article Title: Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space

    doi: 10.1089/ars.2011.3888

    Figure Lengend Snippet: Submitochondrial localization of the UCP3/Trx2 interaction. (A) Mitochondria isolated from HeLa cells transfected with UCP3 and Trx2 were suspended in RIPA buffer+SDS or lysis buffer+Triton X-100, respectively. Mitochondrial pellets and supernatants were subjected to SDS-PAGE followed by immunoblotting (IB) with anti-myc, anti-V5, anti-COX4, and anti-DNA polymerase γ (DPγ) antibodies. PPT, mitochondrial pellet after centrifugation; SDS, RIPA buffer; SUP, supernatant after centrifugation; Triton X-100; lysis buffer. (B) . Mitochondrial pellets and supernatants were resuspended in SDS-PAGE buffer, and then loaded onto an SDS-PAGE gel. Immunoblots show the presence of the intermembrane space (IMS) resident Smac and the matrix resident HSP60 in mitochondria that were untreated or treated with proteinase K (Pro K) (lanes 2–7) and increasing concentrations of digitonin (DIG) (lanes 3–7, 0.1 to 0.7 mg/ml DIG). (C) N (VN)- and C (VC)- terminal fragments of Venus fluorescent proteins were fused to the C-terminus of UCP3 (resides 1–308) (UCP3-VN) or ΔCUCP3 (residues1–234) (ΔCUCP3-VN), and the C-terminus of Trx2, respectively. The fragments of fluorescent proteins of belonging to UCP3-VN and ΔCUCP3-VN were localized to the mitochondrial IMS and matrix, respectively. (D) HeLa cells were transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC. Cell extracts (100 μg) were immunoprecipitated (IP) with anti-myc antibodies and analyzed by IB to detect V5 (UCP3) and myc (Trx2). β-Actin was used as an internal standard. (E) Fluorescent images of HeLa cells transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC as indicated in each panel. (F) ).

    Article Snippet: The proteins were transferred to a nitrocellulose membrane and probed with primary antibody according to the manufacturer's instructions; anti-V5, anti-UCP3, anti-aconitase 2, anti-β-actin (Abcam, Cambridge, MA), anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ), anti-myc, anti-phospho-p38, anti-p38 (Cell Signaling), anti-COX4 (Clontech, Mountain View, CA), anti-DNA polymerase γ (Neo Markers, Fremont, CA), anti-T7 (Novagen, Gibbstown, NJ), and anti-thioredoxin 2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used.

    Techniques: Isolation, Transfection, Lysis, SDS Page, Centrifugation, Western Blot, Immunoprecipitation

    Confronting two-pair primers–polymerase chain reaction for NRF2 -617C/A and -653A/G was carried out at the indicated T a using 2.5U/sample of AmpliTaq Gold polymerase. N: Negative control indicates DNA template omission. MW: Molecular weights (100 bp up to 1000 bp).

    Journal: Future Science OA

    Article Title: Confronting two biomolecular techniques to detect NRF2 gene polymorphism biomarkers

    doi: 10.4155/fsoa-2018-0075

    Figure Lengend Snippet: Confronting two-pair primers–polymerase chain reaction for NRF2 -617C/A and -653A/G was carried out at the indicated T a using 2.5U/sample of AmpliTaq Gold polymerase. N: Negative control indicates DNA template omission. MW: Molecular weights (100 bp up to 1000 bp).

    Article Snippet: All PCRs were performed with 1 unit/sample of Kapa Hot Start Taq polymerase, Biosystems Cat. KK1508 (Sigma-Aldrich, MO, USA) except for the PCR shown in where 2.5 units/sample of AmpliTaq Gold polymerase, Applied Biosystems Cat. N8080161 (ThermoFisher Scientific, MA, USA) were used.

    Techniques: Polymerase Chain Reaction, Negative Control

    The concept of Sequential DEXAS. The outer pair is unlabeled and provided in a 10-fold higher concentration than the inner primers, which are each labeled with different fluorescent dyes. Two thermostable DNA polymerases are used. The Taq DNA polymerase (Taq) is active from the first cycle of the reaction, whereas the AmpliTaq FS DNA polymerase (Taq FS) is activated by a heating step after the first 15 cycles.

    Journal: Nucleic Acids Research

    Article Title: Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction

    doi: 10.1093/nar/gng112

    Figure Lengend Snippet: The concept of Sequential DEXAS. The outer pair is unlabeled and provided in a 10-fold higher concentration than the inner primers, which are each labeled with different fluorescent dyes. Two thermostable DNA polymerases are used. The Taq DNA polymerase (Taq) is active from the first cycle of the reaction, whereas the AmpliTaq FS DNA polymerase (Taq FS) is activated by a heating step after the first 15 cycles.

    Article Snippet: AmpliTaq FS DNA polymerase was purchased from Applied Biosystems (Foster City, CA) and was used in a Sequential DEXAS reaction after a chemical modification.

    Techniques: Concentration Assay, Labeling

    The kinetics of activation of modified AmpliTaq FS. The activity of the chemically modified and not activated AmpliTaq FS DNA polymerase is compared to unmodified AmpliTaq FS DNA polymerase and the modified and activated enzyme.

    Journal: Nucleic Acids Research

    Article Title: Sequential DEXAS: a method for obtaining DNA sequences from genomic DNA and blood in one reaction

    doi: 10.1093/nar/gng112

    Figure Lengend Snippet: The kinetics of activation of modified AmpliTaq FS. The activity of the chemically modified and not activated AmpliTaq FS DNA polymerase is compared to unmodified AmpliTaq FS DNA polymerase and the modified and activated enzyme.

    Article Snippet: AmpliTaq FS DNA polymerase was purchased from Applied Biosystems (Foster City, CA) and was used in a Sequential DEXAS reaction after a chemical modification.

    Techniques: Activation Assay, Modification, Activity Assay

    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: Bsm DNA polymerase, Maxima H Minus Reverse Transcriptase (MHM), Nuclease-Free Water were purchased from Thermo Fisher Scientific.

    Techniques: Incubation, Fluorescence, Agarose Gel Electrophoresis, Marker

    ( a ) Dependence of response (with background subtraction) of modified with Van_74/DP ISFETs to the Bsm DNA polymerase reaction with the addition of different vanillin concentrations. Conditions: mix1 —low molarity selection buffer, 0.2 M ;M dNTP, FP 0.05 pmol/μL, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL; mix2 —low molarity selection buffer, 0.2 M M dNTP, FP 1.0 pmol/μL, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL, T = 22 °C; ( b ) Real time signal of the ISFET (in ∆ϕ) of modified with Van_74/DP ISFETs to the addition of vanillin (final concentration 1 × 10 −8 ) concentration. Conditions: low molarity selection buffer, 0.2 M M dNTP, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Amplified Detection of the Aptamer–Vanillin Complex with the Use of Bsm DNA Polymerase

    doi: 10.3390/s18010049

    Figure Lengend Snippet: ( a ) Dependence of response (with background subtraction) of modified with Van_74/DP ISFETs to the Bsm DNA polymerase reaction with the addition of different vanillin concentrations. Conditions: mix1 —low molarity selection buffer, 0.2 M ;M dNTP, FP 0.05 pmol/μL, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL; mix2 —low molarity selection buffer, 0.2 M M dNTP, FP 1.0 pmol/μL, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL, T = 22 °C; ( b ) Real time signal of the ISFET (in ∆ϕ) of modified with Van_74/DP ISFETs to the addition of vanillin (final concentration 1 × 10 −8 ) concentration. Conditions: low molarity selection buffer, 0.2 M M dNTP, PR 1.6 pmol/μL, Bsm DNA polymerase 0.1 U/μL.

    Article Snippet: Materials Bsm DNA polymerase, large fragment, Bsm buffer, dNTP solution, TBE electrophoresis buffer, and SYBR Gold dye were all purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Modification, Selection, Concentration Assay

    Detection scheme: release of the dehybridized DNA probe during aptamer-vanillin complex formation and the Bsm DNA polymerase reaction with probe detected by ISFET.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Amplified Detection of the Aptamer–Vanillin Complex with the Use of Bsm DNA Polymerase

    doi: 10.3390/s18010049

    Figure Lengend Snippet: Detection scheme: release of the dehybridized DNA probe during aptamer-vanillin complex formation and the Bsm DNA polymerase reaction with probe detected by ISFET.

    Article Snippet: Materials Bsm DNA polymerase, large fragment, Bsm buffer, dNTP solution, TBE electrophoresis buffer, and SYBR Gold dye were all purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques:

    ( a ) Real time signal of the ISFET (in ∆ϕ, with background subtraction) during the Bsm DNA polymerase reaction in homogenous solution in low molarity selection buffer initiated (30–40 s) by the addition of DP at different concentrations (final concentration is marked on the picture); ( b ) Slope (∆ϕ/∆t) dependence on DP concentration calculated from real time signal curves. Reaction conditions: 0.2 mM dNTP, 0.05 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase; ( c ) Real time signal of the ISFET (in ∆ϕ, with background subtraction) during the Bsm DNA polymerase reaction in homogenous solution in low molarity selection buffer initiated (30–40 s) by the addition of DP at different concentrations (final concentration is marked on the picture); ( d ) Slope (∆ϕ/∆t) dependence on DP concentration calculated from real time signal curves. Reaction conditions: 0.2 mM dNTP, 1.0 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Amplified Detection of the Aptamer–Vanillin Complex with the Use of Bsm DNA Polymerase

    doi: 10.3390/s18010049

    Figure Lengend Snippet: ( a ) Real time signal of the ISFET (in ∆ϕ, with background subtraction) during the Bsm DNA polymerase reaction in homogenous solution in low molarity selection buffer initiated (30–40 s) by the addition of DP at different concentrations (final concentration is marked on the picture); ( b ) Slope (∆ϕ/∆t) dependence on DP concentration calculated from real time signal curves. Reaction conditions: 0.2 mM dNTP, 0.05 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase; ( c ) Real time signal of the ISFET (in ∆ϕ, with background subtraction) during the Bsm DNA polymerase reaction in homogenous solution in low molarity selection buffer initiated (30–40 s) by the addition of DP at different concentrations (final concentration is marked on the picture); ( d ) Slope (∆ϕ/∆t) dependence on DP concentration calculated from real time signal curves. Reaction conditions: 0.2 mM dNTP, 1.0 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase.

    Article Snippet: Materials Bsm DNA polymerase, large fragment, Bsm buffer, dNTP solution, TBE electrophoresis buffer, and SYBR Gold dye were all purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques: Selection, Concentration Assay

    Kinetics of Bsm DNA polymerase reaction (after subtraction of the background) monitored by spectrofluorimeter in different buffers and probes. Conditions: buffer, 0.2 mM dNTP, 0.05 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase, 0.1 pmol/μL DP/B1.

    Journal: Sensors (Basel, Switzerland)

    Article Title: Amplified Detection of the Aptamer–Vanillin Complex with the Use of Bsm DNA Polymerase

    doi: 10.3390/s18010049

    Figure Lengend Snippet: Kinetics of Bsm DNA polymerase reaction (after subtraction of the background) monitored by spectrofluorimeter in different buffers and probes. Conditions: buffer, 0.2 mM dNTP, 0.05 pmol/μL FP, 1.6 pmol/μL PR, 0.1 U/μL BSM DNA polymerase, 0.1 pmol/μL DP/B1.

    Article Snippet: Materials Bsm DNA polymerase, large fragment, Bsm buffer, dNTP solution, TBE electrophoresis buffer, and SYBR Gold dye were all purchased from Thermo Fisher Scientific (Waltham, MA, USA).

    Techniques:

    Impact evaluation of Master Mix used in singleplex PCR. In graph: Elizyme = 2X EliZyme HS Robust MIX (Elisabeth Pharmacon, Czech Republic); Accustart II = AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA); Amplitaq = AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA); OneTaq = OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA); Platinum = Platinum Hot Start PCR 2X Master Mix (Invitrogen, California, USA); HotStarTaq = HotStarTaq DNA Polymerase (Qiagen, Germany). YE = Y . enterocolitica; TG = T . gondii ; plus sign in legend = positive sample; minus sign = NTC.

    Journal: Scientific Reports

    Article Title: A novel perspective on MOL-PCR optimization and MAGPIX analysis of in-house multiplex foodborne pathogens detection assay

    doi: 10.1038/s41598-019-40035-5

    Figure Lengend Snippet: Impact evaluation of Master Mix used in singleplex PCR. In graph: Elizyme = 2X EliZyme HS Robust MIX (Elisabeth Pharmacon, Czech Republic); Accustart II = AccuStart II PCR ToughMix (QuantaBio, Massachusetts, USA); Amplitaq = AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific, Massachusetts, USA); OneTaq = OneTaq Hot Start 2X Master Mix with GC Buffer (New England BioLabs, Massachusetts, USA); Platinum = Platinum Hot Start PCR 2X Master Mix (Invitrogen, California, USA); HotStarTaq = HotStarTaq DNA Polymerase (Qiagen, Germany). YE = Y . enterocolitica; TG = T . gondii ; plus sign in legend = positive sample; minus sign = NTC.

    Article Snippet: In order to find a balance, the performance of six different master mixes was tested (Fig. ), including the previously utilized HotStarTaq DNA Polymerase – and the upgraded AmpliTaq Gold 360 Master Mix , .

    Techniques: Polymerase Chain Reaction, Hot Start PCR

    Multiplex TETR-PCR products obtained for Chlamydia species with species-specific primer sets CTR 70/71, CPN 90/91, and CPS 100/101 targeting the 16S and 16S-23S spacer rRNA genes. One IFU per PCR was tested i ndividually and in combinations. Base-pair sizes of weight markers (wm) and PCR products are indicated in the margins. DNA from C. trachomatis VR 348 serovar E, C. pneumoniae BAL 37, and C. psittaci SM006 was amplified.

    Journal: Journal of Clinical Microbiology

    Article Title: Touchdown Enzyme Time Release-PCR for Detection and Identification of Chlamydia trachomatis, C. pneumoniae, and C. psittaci Using the 16S and 16S-23S Spacer rRNA Genes

    doi:

    Figure Lengend Snippet: Multiplex TETR-PCR products obtained for Chlamydia species with species-specific primer sets CTR 70/71, CPN 90/91, and CPS 100/101 targeting the 16S and 16S-23S spacer rRNA genes. One IFU per PCR was tested i ndividually and in combinations. Base-pair sizes of weight markers (wm) and PCR products are indicated in the margins. DNA from C. trachomatis VR 348 serovar E, C. pneumoniae BAL 37, and C. psittaci SM006 was amplified.

    Article Snippet: In addition, analytical sensitivity with spiked clinical specimens were tested by use of the TETR-PCR protocol described above but with an alternative DNA polymerase (2 U of HotStarTaq DNA polymerase; Qiagen, Valencia, Calif.) and 1.5 mM MgCl2 .

    Techniques: Multiplex Assay, Polymerase Chain Reaction, Amplification

    Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Plasmid DNA from the cecal sample after amplification with phi29 polymerase. 1 , 1 kb ladder and 2 , Plasmid DNA amplified with Phi29 DNA polymerase.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Amplification

    Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Journal: Frontiers in Microbiology

    Article Title: A Comparison of Methods for the Extraction of Plasmids Capable of Conferring Antibiotic Resistance in a Human Pathogen From Complex Broiler Cecal Samples

    doi: 10.3389/fmicb.2018.01731

    Figure Lengend Snippet: Digested plasmid DNA extracted from E. coli transformants after electroporation with the phi29 polymerase amplified DNA. 1 , 1 kb ladder; Plasmid DNA extracted from transformants selected on agar plates containing; 2 , ampicillin 32 mg/L (M_Amp_BC); 3 , ampicillin 32 mg/L (M_Amp_SC); 4 , tetracycline 16 mg/L (M_Tet_BC); 5 , tetracycline 16 mg/L (M_Tet_SC); 6 , kanamycin 25 mg/L (M_Kan); 7 , ciprofloxacin 16 mg/L (M_Cip). BC and SC refer to the two different colony morphology types, big or small colonies, on the same antibiotic plate.

    Article Snippet: Samples were incubated at 95°C for 5 min and immediately chilled on ice for 5 min. 1.6 μL phi29 DNA polymerase (New England Biolabs), 0.02 μL of inorganic pyrophosphatase (from yeast) (New England Biolabs) and 2 μL of dNTPs (10 mM) (Thermo Scientific) were added and incubated at 30°C for 16 h. Amplified plasmid DNA (5 μL) was electroporated at 1.8 kV into 15 μL of E. coli DH5α cells.

    Techniques: Plasmid Preparation, Electroporation, Amplification

    Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Effect of RNA substitutions in circular templates on rolling circle amplification with phi29 DNA polymerase. ( A ) Total amount of RCA products (y-axis) generated for padlock probes with/without a terminal 3′ RNA and in the absence of synthetic RNA ligation template (template -). ( B ) Circles with 0–7 RNA substitutions in the backbone were amplified and digitally counted. The y-axis shows the number of rolling circle products (RCPs); error bars ± S.D.; n = 2. The same RCA reactions with chimeric circles were also monitored in real-time by measuring SYBR Gold incorporation on qPCR instrument ( C and E ). (C) RCA reaction curves of circles with 0, 1 and 2 RNA substitutions. ( D ) RCPs from C were imaged on microscope slides and size and intensity of individual RCPs were quantified. Black line, median; upper whisker, highest value that is within 1.5 the interquartile range of the hinge; lower whisker, lowest value within 1.5 the interquartile range of the hinge. (E) Real-time data of the same RCA reactions as in B with 0–7 RNA substitutes are displayed. Representative samples are presented from a duplicated experiment. To highlight the initial stages of RCA and to show the difference between the samples with low RCA efficiency, fluorescence intensity readout between 3000 and 6000 is presented.

    Article Snippet: Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C.

    Techniques: Amplification, Generated, Ligation, Real-time Polymerase Chain Reaction, Microscopy, Whisker Assay, Fluorescence

    Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: Phi29 DNA polymerase exhibits higher RCA rate with circles containing pyrimidine RNA substitutions. ( A ) Real-time RCA curves of circles containing 1, 2, 3 or 4 consecutive RNA substations of rG, rU, rA, rC RNA bases are displayed (number of consecutive substitutions is indicated above plots). Rate of RCA was monitored by measuring fluorescence build-up (y-axis) resulted from SYBR Gold incorporation into RCPs. Averaged fluorescence intensity for each RCA time point was calculated from a duplicated experiment. RCA was conducted in the presence of Mg 2+ and Mn 2+ (solid and dashed lines respectively). ( B ) Linear, early stage RCA velocity (y-axis) is presented for PLPs from (A) in the presence of Mg 2+ (solid lines) and Mn 2+ (dashed lines). ( C ) RCA for the control PLP (non-chimeric DNA circle, with Mg 2+ (solid) and Mn 2+ (dashed line) are displayed.

    Article Snippet: Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C.

    Techniques: Fluorescence, Plasmid Purification

    DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Journal: Nucleic Acids Research

    Article Title: Limited reverse transcriptase activity of phi29 DNA polymerase

    doi: 10.1093/nar/gky190

    Figure Lengend Snippet: DNA sequencing-based analysis of rolling circle products reveals reverse transcription activity of phi29 DNA polymerase. ( A ) After RCA, short DNA oligonucleotides were hybridized to an AluI restriction site in the RCA products and RCPs were digested with AluI restriction enzyme, resulting in RCA monomers. Following digestion, monomers were PCR-amplified using primers containing Ilumina adapter sequences. PCR products were extended using IIlumina indexed primers. Finally, sequencing library was prepared using indexed primers-specific P5/7 PCR primers. The region of interest containing RNA substitutions in the original padlock probe sequence is indicated with green boxes. ( B ) Logos showing sequencing frequencies for each position within RCA monomers generated from the control DNA circle (P1 = dG), and circles containing single rG, rU, rA and rC substitutions at the RNA position (P1). Positions P1 and P2 are indicated and position P1 was additionally highlighted with the red box. ( C ) Incorporation of incorrect nucleotides for every position in the sequenced monomers from (B). Error rates, calculated as Incorporation error [%] = 1 – number of reads with expected nucleotide/total number of reads, is presented for padlock probes with single- (upper plot) and double-RNA substitutions (lower plots). P1 position for the first RNA substitution is indicated with the box.

    Article Snippet: Next, RCA products were digested with AluI restriction enzyme in a reaction mixture containing 1 × phi29 DNA polymerase buffer, 0.2 μg/μl BSA, 100 nM restriction oligonucleotide , 120 mU/μl AluI (NEB) and RCA products at a final concentration of 10 pM during 10 min incubation at 37°C.

    Techniques: DNA Sequencing, Activity Assay, Polymerase Chain Reaction, Amplification, Sequencing, Generated

    Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Journal: mBio

    Article Title: Selective Whole-Genome Amplification Is a Robust Method That Enables Scalable Whole-Genome Sequencing of Plasmodium vivax from Unprocessed Clinical Samples

    doi: 10.1128/mBio.02257-16

    Figure Lengend Snippet: Selective whole-genome amplification (SWGA) of Plasmodium vivax genomic DNA (gDNA) from human blood samples. (A) SWGA primers bind frequently to Plasmodium vivax gDNA and infrequently to human gDNA. (B) When phi29 encounters double-stranded gDNA, it displaces the newly synthesized strand, opening new primer binding sites on the synthesized gDNA, leading to selective amplification of templates with frequent primer binding sites. (C) Post-SWGA, the percentage of P. vivax DNA has increased relative to the percentage of host DNA.

    Article Snippet: Thirty to 70 ng of input DNA was added to a 50-µl reaction mixture containing 3.5 µM SWGA primers, 30 U phi29 DNA polymerase enzyme (New England Biolabs), 1× phi29 buffer (New England Biolabs), 4 mM dNTPs (Roche), 1% bovine serum albumin, and water.

    Techniques: Whole Genome Amplification, Synthesized, Binding Assay, Amplification

    Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by T4 DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P

    Journal: Genome Research

    Article Title: Guide Positioning Sequencing identifies aberrant DNA methylation patterns that alter cell identity and tumor-immune surveillance networks

    doi: 10.1101/gr.240606.118

    Figure Lengend Snippet: Guide Positioning Sequencing (GPS) detects genome-wide DNA methylation accurately with high coverage rate. ( A ) Schematic of GPS workflow for DNA methylation detection. The gray line represents original DNA sequence, and the orange line represents DNA treated by T4 DNA polymerase, which replaces cytosine with 5′-methylcytosine at 3′ end of DNA fragment. The solid circle (●) represents methylated cytosine, and the open circle (○) represents unmethylated cytosine, whereas the triangle (Δ) represents thymine. Blue and green short lines represent the NGS linker. Read1 represents the bisulfite-converted 5′ end of fragments, whereas Read2 represents the 3′ end of fragments, which is the same as the genome sequence due to 5′-methylcytosine replacement. ( B ) The accurate alignment rate of Bowtie 2 and GPS is obviously higher than that in BSMAP based on simulated data: (***) P

    Article Snippet: Thirty units of T4 DNA polymerase (New England BioLabs, M0203L) was used to perform 3′→5′ digestion of the DNA fragments for 100 min at 12°C followed by adding 10 µL dNTP mix which contained dATP, dTTP, dGTP, and 5′-methyl-dCTP nucleotide (final concentration 0.5 mM) and incubating for 30 min at 37°C.

    Techniques: Sequencing, Genome Wide, DNA Methylation Assay, Methylation, Next-Generation Sequencing

    Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Test of QC cloning using Klenow DNA polymerase. (A) Test of Klenow exonuclease activity determined using the same assay used for T4 DNA polymerase. (B) To test QC cloning using Klenow DNA polymerase, the PCR product T019 GC3F was cloned into pICH31477 (23 nucleotide catching sequence) and pICH31480 (52 nucleotide catching sequence). Incubation was performed at 37°C for 0, 30, 60, 90, and 120 minutes. ( C ) Eight randomly chosen clones from 120 min time points were analyzed by colony PCR using vector primers. The size of the expected full-length fragment is indicated by an arrow.

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Clone Assay, Activity Assay, Polymerase Chain Reaction, Sequencing, Incubation, Plasmid Preparation

    Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Strategy for amplification and QC cloning of immunoglobulin fragments. ( A ) Amplification of immunoglobulin fragments from non-Hodgkin lymphoma samples. Total RNA extracted from biopsy samples (1) is reverse-transcribed into first strand cDNA using an oligo dT primer (2). The cDNA is column-purified to remove remaining dNTPs, and G-tailed using terminal transferase and dGTP (3). (4) The G-tailed cDNA is used as a template for PCR amplification using a G-tail adaptor primer (bap2 pc) and an immunoglobulin constant region-specific primer (gsp). The PCR product is column-purified to remove the remaining dNTPs (5). ( B ) Preparation of vector for QC cloning. The cloning vector is linearized using the enzyme Pst I. ( C ) The column-purified PCR product and the linearized vector are mixed and treated with T4 DNA polymerase to generate single-stranded ends that are complementary between the vector and insert (7). The mixture is directly transformed into chemo-competent E. coli DH10B cells where the annealed ends of the vector and insert complex are repaired and ligated (8). (9) After cloning, the plasmid is purified and the insert sequenced using a vector specific primer (seqpr).

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Amplification, Clone Assay, Purification, Polymerase Chain Reaction, Plasmid Preparation, Transformation Assay

    Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Test of QC cloning performed with or without heat inactivation. ( A ) PCR product amplified from G-tailed cDNA prepared from biopsy sample T019 using primers bap2 pc and GC3F. ( B ) Structure of the vector and of the PCR product. ( C , D ) The PCR product was cloned into pICH31480 using T4 DNA polymerase treatment for 5 minutes at 25°C (A, adaptor; U, unknown sequence; K, known sequence; CS, catching sequence), followed by heat inactivation 20 min at 75°C ( C ) or incubation at 4°C ( D ). Eight randomly chosen clones were analyzed by colony PCR using vector primers. The products amplified by colony PCR were separated on a 1% agarose gel supplemented with ethidium bromide and visualized under UV light. The expected insert size is indicated by an arrow.

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Incubation, Agarose Gel Electrophoresis

    Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Journal: PLoS ONE

    Article Title: Quick and Clean Cloning: A Ligation-Independent Cloning Strategy for Selective Cloning of Specific PCR Products from Non-Specific Mixes

    doi: 10.1371/journal.pone.0020556

    Figure Lengend Snippet: Quantification of T4 DNA polymerase exonuclease activity. Sac II/ Nde I-digested plasmid DNA (3 fragments, lane C) was treated with T4 DNA polymerase for 10 minutes at 25°C, 20°C, 15°C and 10°C. The T4 DNA polymerase was then inactivated by incubation at 80°C for 5 min. The single-stranded ends generated by the 3′ to 5′ exonuclease activity T4 DNA polymerase were removed by using Mung Bean nuclease. The size of the resulting fragments was analyzed by agarose gel electrophoresis. As a control for the heat inactivation of T4 DNA polymerase, digested plasmid DNA was inactivated at 80°C for 5 minutes immediately after addition of T4 DNA polymerase (lane H).

    Article Snippet: To perform the QC cloning 2 µl PCR product, 1 µl Bpi I-digested vector, 2 µl 10x T4 DNA polymerase buffer, 0.5 µl T4 DNA polymerase (New England Biolabs, Ipswich MA, USA; 3 units/ µl) and 14.5 µl water were mixed and incubated for 5 minutes at room temperature.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Generated, Agarose Gel Electrophoresis

    The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Journal: Nucleic Acids Research

    Article Title: A novel and simple method for construction of recombinant adenoviruses

    doi: 10.1093/nar/gkl449

    Figure Lengend Snippet: The PCR product of a foreign gene was amplified by T4 DNA polymerase and dGTP, and then was ligated with the Bsu36I-digested pRTRA. The ligation mixture was transformed to the donor strain DH10β, and then the recombinant donor plasmid was obtained. We introduced the two different Bsu36I sites (CCTTAGG and CCTGAGG) in the pRTRA vector and the 4 nt TTAC(5′–3′) in the forward primer and the other 4 nt TGAC(5′–3′) in the reverse primer. The complete digestion of pRTRA with Bsu36I results in a linearized donor vector with overhang ends of 5′-TTA-3′ and 5′-TCA-3′, respectively. We made use of the 3′→5′ exonuclease activity and 5′→3′ polymerase activity of T4 DNA polymerase. When T4 DNA polymerase encounters the first Guanine nucleotide at the 5′ end of the DNA in the dGTP bath, the reaction will keep the balance between the exonuclease activity and polymerase activity. Therefore, the overhang ends of the gene fragments of interest will be digested to be perfectly compatible with the vector.

    Article Snippet: Cloning the foreign genes gfp and man into the donor plasmid using restriction enzyme Bsu36I and T4 DNA polymerase The gfp gene was amplified from pEGFP-1 (Clontech) by PCR.

    Techniques: Polymerase Chain Reaction, Amplification, Ligation, Transformation Assay, Recombinant, Plasmid Preparation, Activity Assay

    TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a DNA template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, Therminator, 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.

    Journal: Journal of the American Chemical Society

    Article Title: Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

    doi: 10.1021/ja0428255

    Figure Lengend Snippet: TNA polymerase screen. Products of tNTP elongation on a 5‘[-P 32 ]-labeled 23-mer primer annealed to a DNA template. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for experiments using exonuclease deficient family B DNA polymerases: 9°N, Therminator, 9°N single mutant Y409V, 9°N double mutant Y409V and A485L, Deep Vent, and Vent. Time points were taken for each polymerase reaction at 0 (no enzyme), 15, 30, 90, 150, and 300 min, lanes 1−6, respectively.

    Article Snippet: Polymerization reactions were initiated by adding 10 μL of 2 × dNTP or tNTP solution (0.01−10 μM) to an equal volume of the reaction mixture containing primer−template complex, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM MgSO4 , 0.1% Triton X-100, 0.25 μg/μL BSA, 100 μM DTT, and either 0.05 units of Therminator DNA polymerase (final concentration 9.3 nM) (New England Biolabs, 2 u/μL) or 0.1 units of Deep Vent (exo-) DNA polymerase (final concentration 1.82 nM) (New England Biolabs, 2 u/μL).

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Mutagenesis

    TNA primer extension reactions. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for (A) Deep Vent exo- (DV exo- ) and (B) Therminator-catalyzed DNA polymerase reactions. Primer-extension reactions were performed in the absence (lanes 1−7) and presence (lanes 8−14) of 1.25 mM MnCl 2 .

    Journal: Journal of the American Chemical Society

    Article Title: Kinetic Analysis of an Efficient DNA-Dependent TNA Polymerase

    doi: 10.1021/ja0428255

    Figure Lengend Snippet: TNA primer extension reactions. Reaction progress over time was analyzed by denaturing polyacrylamide gel electrophoresis for (A) Deep Vent exo- (DV exo- ) and (B) Therminator-catalyzed DNA polymerase reactions. Primer-extension reactions were performed in the absence (lanes 1−7) and presence (lanes 8−14) of 1.25 mM MnCl 2 .

    Article Snippet: Polymerization reactions were initiated by adding 10 μL of 2 × dNTP or tNTP solution (0.01−10 μM) to an equal volume of the reaction mixture containing primer−template complex, 20 mM Tris-HCl, 10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM MgSO4 , 0.1% Triton X-100, 0.25 μg/μL BSA, 100 μM DTT, and either 0.05 units of Therminator DNA polymerase (final concentration 9.3 nM) (New England Biolabs, 2 u/μL) or 0.1 units of Deep Vent (exo-) DNA polymerase (final concentration 1.82 nM) (New England Biolabs, 2 u/μL).

    Techniques: Polyacrylamide Gel Electrophoresis