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  • 99
    New England Biolabs taq1 dna polymerase
    Taq1 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq1 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    taq1 dna polymerase - by Bioz Stars, 2020-08
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    99
    Thermo Fisher taq dna polymerase
    A mobility shift assay for TaqS <t>DNA</t> polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- <t>Taq</t> S DNA polymerase, respectively.
    Taq Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 44901 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 44901 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2020-08
    99/100 stars
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    99
    Qiagen taq dna polymerase
    Slowly migrating DNAs are converted into ocDNA by <t>Taq</t> (A) or T4 (B) <t>DNA</t> polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.
    Taq Dna Polymerase, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 9377 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/Qiagen
    Average 99 stars, based on 9377 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2020-08
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    95
    Promega taq polymerase
    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt <t>PCR</t> fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for <t>Taq</t> polymerase during the PCR reaction but 8c is not.
    Taq Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 16516 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/Promega
    Average 95 stars, based on 16516 article reviews
    Price from $9.99 to $1999.99
    taq polymerase - by Bioz Stars, 2020-08
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    99
    TaKaRa taq dna polymerase
    Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by <t>Taq</t> <t>DNA</t> polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.
    Taq Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 13199 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/TaKaRa
    Average 99 stars, based on 13199 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2020-08
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    Image Search Results


    A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: A mobility shift assay for TaqS DNA polymerase (A) and NeqSSB-TaqS DNA polymerase (B) with ssDNA and dsDNA. The output products were analyzed on a 2% agarose gel with ethidium bromide in the UV light. The reaction mix contained 10 pmol Oligo (dT) 76 and/or 2.5 pmol PCR product with a length of 100 bp. In panel A: 1. Oligo (dT) 76 and 0 pmol TaqS DNA polymerase; 2. 100 bp PCR product and 0 pmol DNA polymerase; 3–9. Oligo (dT) 76 and 100 bp PCR product with 24,6; 49,2; 98,4; 196,8; 393,6; 787,2; 1574,4 pmol of TaqS DNA polymerase, respectively. In panel B: 11. Oligo (dT) 76 and 0 pmol NeqSSB-TaqS DNA polymerase. 12. 100 bp PCR product and 0 pmol NeqSSB-TaqS DNA polymerase. 13–19. Oligo (dT) 76 and 100 bp PCR product with 3,3; 6,6; 13,2; 26,4; 52,8; 105,6; 211,2 pmol Neq SSB- Taq S DNA polymerase, respectively.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Mobility Shift, Agarose Gel Electrophoresis, Polymerase Chain Reaction

    Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Evaluation of PCR amplification rate. Comparison of the PCR amplification rates of a fusion Neq SSB -TaqS DNA polymerase for 300 bp (A), 500 bp (B), 1000 bp (C) products and a Taq S DNA polymerase for 300 bp (D), 500 bp (E), 1000 bp (F) products. The elongation times used for the PCR amplification are indicated at the top. Lane M: the DNA molecular size marker (50–2000 bp).

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Polymerase Chain Reaction, Amplification, Marker

    Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Journal: PLoS ONE

    Article Title: Fusion of Taq DNA polymerase with single-stranded DNA binding-like protein of Nanoarchaeum equitans—Expression and characterization

    doi: 10.1371/journal.pone.0184162

    Figure Lengend Snippet: Characterization of a fusion Neq SSB -TaqS DNA polymerase in comparison to a Taq S DNA polymerase. The effect of (A) MgCl 2 , (B) KCl, (C) (NH 4 ) 2 SO 4 , (D) pH and (E) temperature on the polymerase activity. The results for the NeqSSB-TaqS DNA polymerase are marked with black circles, whilst for the Taq S DNA polymerase with black tringles. Error bars for the TaqS DNA polymerase have the end bar whilst for the Neq - Taq S DNA polymerase does not have the end bar.

    Article Snippet: The processivity values determined with the use of a heparin trap were much lower than the values previously published for the Taq DNA polymerase [ , ] and were within a range of 80 nt to 160 nt.

    Techniques: Activity Assay

    Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Journal: Journal of Virology

    Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

    doi: 10.1128/JVI.75.22.10573-10581.2001

    Figure Lengend Snippet: Slowly migrating DNAs are converted into ocDNA by Taq (A) or T4 (B) DNA polymerase treatment. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slowly migrating viral DNAs are indicated with an asterisk (∗). (A) TNAs were extracted from wt protoplasts at 72 h posttransfection with pTOM6 alone (the two lanes on the right) or together with pTOM100C4(−) or pTOM100NT and analyzed directly (−) or following incubation with Taq DNA polymerase for the time indicated below. (B) TNAs were extracted from wt or transgenic (102.22) protoplasts at 72 h posttransfection with pSP97 (TYLCSV-ES[1]) and analyzed following a 1-h incubation with (+) or without (−) T4 DNA polymerase. Lane C, TNAs from a TYLCSV-infected tomato plant digested with Bgl II to show migration of linear DNA.

    Article Snippet: To verify the nature of these slowly migrating molecules, TNAs extracted from the samples at 24 and 72 h were incubated with Taq DNA polymerase and analyzed by Southern blot (Fig. B).

    Techniques: Incubation, Transgenic Assay, Infection, Migration

    Slowly migrating DNAs also accumulate at early stages after transfection of wt protoplasts with TYLCSV. TNAs were extracted from protoplasts transfected with pTOM6. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slow-migrating viral DNAs are indicated with an asterisk (∗). HPT, hours posttransfection. (A) Time course analysis of viral DNA forms. Sample at 72 h was diluted 20-fold to give signals of satisfactory intensity on the autoradiographic film. (B) Taq DNA polymerase treatment of TNAs at 24 and 72 h after transfection. Samples were incubated for 10 min at 37°C with (+) or without (−) the polymerase. Lane C, sample at 72 h digested with Bgl II to show the linear DNA.

    Journal: Journal of Virology

    Article Title: Transgenically Expressed T-Rep of Tomato Yellow Leaf Curl Sardinia Virus Acts as a trans-Dominant-Negative Mutant, Inhibiting Viral Transcription and Replication

    doi: 10.1128/JVI.75.22.10573-10581.2001

    Figure Lengend Snippet: Slowly migrating DNAs also accumulate at early stages after transfection of wt protoplasts with TYLCSV. TNAs were extracted from protoplasts transfected with pTOM6. The blots were hybridized with a C1-sense RNA probe. The positions of ocDNA, linear DNA (linDNA), scDNA, and cssDNA forms of viral DNA are indicated. Slow-migrating viral DNAs are indicated with an asterisk (∗). HPT, hours posttransfection. (A) Time course analysis of viral DNA forms. Sample at 72 h was diluted 20-fold to give signals of satisfactory intensity on the autoradiographic film. (B) Taq DNA polymerase treatment of TNAs at 24 and 72 h after transfection. Samples were incubated for 10 min at 37°C with (+) or without (−) the polymerase. Lane C, sample at 72 h digested with Bgl II to show the linear DNA.

    Article Snippet: To verify the nature of these slowly migrating molecules, TNAs extracted from the samples at 24 and 72 h were incubated with Taq DNA polymerase and analyzed by Southern blot (Fig. B).

    Techniques: Transfection, Incubation

    Optimisation of the PPLMD method . The y-axis represents the average background-subtracted pixel density (0–64,000). A. Introduction of SpikeTarget DNA reduced background in negative control (purple vs. yellow). Blue: genomic DNA (containing RRS and MON810) + SpikeTarget, purple: MQ, yellow: MQ with SpikeTarget. All 4 padlock probes were present in the mix. HotStar Taq DNA polymerase was used to amplify in 40 LATE cycles. B. 80 LATE cycles increased signal compared to 40 cycles. Purple: 40 LATE cycles, blue: 80 LATE cycles. All 4 padlock probes were present in the mix (containing RRS and MON810). Vent ® exo - DNA polymerase was used.

    Journal: BMC Genomics

    Article Title: Optimised padlock probe ligation and microarray detection of multiple (non-authorised) GMOs in a single reaction

    doi: 10.1186/1471-2164-9-584

    Figure Lengend Snippet: Optimisation of the PPLMD method . The y-axis represents the average background-subtracted pixel density (0–64,000). A. Introduction of SpikeTarget DNA reduced background in negative control (purple vs. yellow). Blue: genomic DNA (containing RRS and MON810) + SpikeTarget, purple: MQ, yellow: MQ with SpikeTarget. All 4 padlock probes were present in the mix. HotStar Taq DNA polymerase was used to amplify in 40 LATE cycles. B. 80 LATE cycles increased signal compared to 40 cycles. Purple: 40 LATE cycles, blue: 80 LATE cycles. All 4 padlock probes were present in the mix (containing RRS and MON810). Vent ® exo - DNA polymerase was used.

    Article Snippet: The results showed a tendency that positive signals were improved and background signals were decreased compared to the use of HotStar Taq DNA polymerase, although statistical evidence is lacking (results not shown).

    Techniques: Negative Control

    Effect of individual constituents of the PCR on background fluorescence in the presence or absence of CABS. Each column represents the result of three PCRs performed: 1, in the absence of Taq Gold polymerase, but in the presence of oligonucleotides and template; 2, in the absence of Taq Gold polymerase and template, but in the presence of oligonucleotides; 3, in the absence of Taq Gold polymerase, template, and one oligonucleotide but in the presence of one oligonucleotide; 4, in the absence of primers and template, but in the presence of Taq Gold polymerase; 5, in the absence of template, but in the presence of both primers and polymerase; 6, as in case 3, except in the presence of the alternative primer of the primer combination; 7, in which specific amplicon is produced; and 8, in which no amplicon is expected. Conditions 1, 2, and 6 show the effect of CABS in reducing the background fluorescence caused by the single-strand oligonucleotides. The reaction with CABS in conditions 5 and 8 shows the residual effect caused by primer/dimers, in the case of 8 with additional template DNA.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Alkaline-mediated differential interaction (AMDI): A simple automatable single-nucleotide polymorphism assay

    doi: 10.1073/pnas.041619998

    Figure Lengend Snippet: Effect of individual constituents of the PCR on background fluorescence in the presence or absence of CABS. Each column represents the result of three PCRs performed: 1, in the absence of Taq Gold polymerase, but in the presence of oligonucleotides and template; 2, in the absence of Taq Gold polymerase and template, but in the presence of oligonucleotides; 3, in the absence of Taq Gold polymerase, template, and one oligonucleotide but in the presence of one oligonucleotide; 4, in the absence of primers and template, but in the presence of Taq Gold polymerase; 5, in the absence of template, but in the presence of both primers and polymerase; 6, as in case 3, except in the presence of the alternative primer of the primer combination; 7, in which specific amplicon is produced; and 8, in which no amplicon is expected. Conditions 1, 2, and 6 show the effect of CABS in reducing the background fluorescence caused by the single-strand oligonucleotides. The reaction with CABS in conditions 5 and 8 shows the residual effect caused by primer/dimers, in the case of 8 with additional template DNA.

    Article Snippet: The reaction mixture consisted of 67 mM Tris⋅HCl (pH 8.4) at 25°C, 16.6 mM ammonium sulfate, 2 mM magnesium chloride, 0.01% (vol/vol) Tween 20, 200 μM of each dNTP, 1 μM of each primer, and 0.5 units Hotstart Taq DNA polymerase (Qiagen) or AmpliTaq Gold (Perkin–Elmer).

    Techniques: Polymerase Chain Reaction, Fluorescence, Amplification, Produced

    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

    Journal: Nucleic Acids Research

    Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

    doi:

    Figure Lengend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5c – 8c , derived from urocanic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6c ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5c ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8c ; lane 7, molecular weight markers. Triphosphates 5c and 6c (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8c is not.

    Article Snippet: Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

    PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

    Journal: Nucleic Acids Research

    Article Title: Enhancing the catalytic repertoire of nucleic acids: a systematic study of linker length and rigidity

    doi:

    Figure Lengend Snippet: PAGE gel image of the ethidium bromide stained and UV visualised 98 nt PCR fragment using pUC19 as template and the C5-imidazole-modified triphosphates 5b – 8b , derived from imidazole 4-acetic acid. The full experimental conditions are given in Materials and Methods. Lane 1, molecular weight markers; lane 2, a PCR reaction containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR reaction containing dATP, dCTP and dGTP, which does not result in formation of any product; lane 4, a PCR reaction containing dATP, dCTP, dGTP and 6b ; lane 5, a PCR reaction containing dATP, dCTP, dGTP and 5b ; lane 6, a PCR reaction containing dATP, dCTP, dGTP and 8b ; lane 7, molecular weight markers. Triphosphates 5b and 6b (Fig. 2) are substrates for Taq polymerase during the PCR reaction but 8b is not.

    Article Snippet: Consequently, we have studied the incorporation of the modified triphosphates during PCR reactions mediated by Taq polymerase using template DNAs varying from 62 to 224 nt.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Modification, Derivative Assay, Molecular Weight

    Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.

    Journal: PLoS ONE

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    doi: 10.1371/journal.pone.0123468

    Figure Lengend Snippet: Blocking efficiency of three kinds of blocked primers in PCR amplifications mediated by Taq DNA polymerase and high-fidelity DNA polymerase. (a) Primer and template sets. WT and Mu indicate wild-type and mutant types, respectively. The 3'-blocked forward primer completely matches the WT sequence but forms a mismatch with the Mu sequence (Mu*1) at the 3'-end. The reverse primer matches both the WT and Mu sequences. The 3'-terminal nucleotide of the blocked primer is blocked with-Pi or-Amino C6, or replaced with ddCTP. (b) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by Taq DNA polymerase. (c) Blocking efficiency of-Pi,-Amino C6 or-ddC in PCR mediated by high-fidelity DNA polymerase (KOD FX). (d) Discrimination efficiency of the typical proofreading-PCR medicated by various amounts (0.5, 1, 1.5 and 2 U) of KOD FX DNA polymerase using the ddC-blocked primer. All PCR amplifications were performed in a total volume of 20 μL containing 1 U of Taq or KOD FX DNA polymerase (Panel b and c) or various amounts of KOD FX DNA polymerase (Panel d). The cycling conditions consisted of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. NTC: no-template control.

    Article Snippet: The results showed that the use of 0.1–0.15 U of high-fidelity DNA polymerase combined with 1 U of Taq DNA polymerase in per 20 μL reaction resulted in the best discrimination and amplification effects ( ).

    Techniques: Blocking Assay, Polymerase Chain Reaction, Mutagenesis, Sequencing

    Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.

    Journal: PLoS ONE

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    doi: 10.1371/journal.pone.0123468

    Figure Lengend Snippet: Optimization of annealing temperature and amount of high-fidelity DNA polymerase in the modified PR-PCR. (a) Discrimination efficiency of the modified PR-PCR for known mutations using a mixture of 1 U of Taq DNA polymerase and various amount (0.05, 0.1 and 0.3 U) of KOD FX DNA polymerase in the reaction. The PCR amplifications were performed in a total volume of 20 μL under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 56°C for 20 s and extension at 72°C for 25 s. (b) Optimization of annealing temperature for the modified PR-PCR using a mixture of 1 U of Taq DNA polymerase and 0.1 U of KOD FX DNA polymerase. As the optimal discrimination efficiency was obtained when the annealing temperature was within a scope of 61.8°C–62.6°C, the results from 50°C to 61.2°C were not shown. The temperature 62.2°C was selected for subsequent experiments. (c) Optimization of amount of high-fidelity DNA polymerase for the modified PR-PCR with an input of 1 U of Taq DNA polymerase at an annealing temperature of 62.2°C.

    Article Snippet: The results showed that the use of 0.1–0.15 U of high-fidelity DNA polymerase combined with 1 U of Taq DNA polymerase in per 20 μL reaction resulted in the best discrimination and amplification effects ( ).

    Techniques: Modification, Polymerase Chain Reaction

    Sensitivity and selectivity of the modified PR-PCR for mutation detection using a fusion-blocked primer and adaptor. (a) Diagram of the fusion-blocked primer and adaptor used in the reactions. (b) Primer and adaptor sequences. (c) Sensitivity of the modified PR-PCR. (d) Selectivity of the modified PR-PCR. All reactions were performed in a total volume of 25 μL containing a mixture of 1 μL of gDNA template at 50 ng/μL, 2.5 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 0.15 U of PrimeSTAR HS DNA polymerase, 0.75 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of the reverse primer, fusion-blocked forward primer and adaptor. The DNA templates were prepared by mixing different amounts of MCF-7 (WT) gDNA (from 0 to 50 ng) among HCC1937 (mutant type, MT) gDNA (from 50 to 0 ng) with concentrations from 0 to 100%. The reactions were performed under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 30 s and extension at 72°C for 20 s.

    Journal: PLoS ONE

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer

    doi: 10.1371/journal.pone.0123468

    Figure Lengend Snippet: Sensitivity and selectivity of the modified PR-PCR for mutation detection using a fusion-blocked primer and adaptor. (a) Diagram of the fusion-blocked primer and adaptor used in the reactions. (b) Primer and adaptor sequences. (c) Sensitivity of the modified PR-PCR. (d) Selectivity of the modified PR-PCR. All reactions were performed in a total volume of 25 μL containing a mixture of 1 μL of gDNA template at 50 ng/μL, 2.5 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 0.15 U of PrimeSTAR HS DNA polymerase, 0.75 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of the reverse primer, fusion-blocked forward primer and adaptor. The DNA templates were prepared by mixing different amounts of MCF-7 (WT) gDNA (from 0 to 50 ng) among HCC1937 (mutant type, MT) gDNA (from 50 to 0 ng) with concentrations from 0 to 100%. The reactions were performed under cycling conditions consisting of pre-denaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 30 s and extension at 72°C for 20 s.

    Article Snippet: The results showed that the use of 0.1–0.15 U of high-fidelity DNA polymerase combined with 1 U of Taq DNA polymerase in per 20 μL reaction resulted in the best discrimination and amplification effects ( ).

    Techniques: Modification, Polymerase Chain Reaction, Mutagenesis