taq dna polymerase  (TaKaRa)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    Name:
    LA Taq polymerase
    Description:

    Catalog Number:
    LTP1360476
    Price:
    None
    Score:
    85
    Buy from Supplier


    Structured Review

    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.

    https://www.bioz.com/result/taq dna polymerase/product/TaKaRa
    Average 97 stars, based on 215 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-12
    97/100 stars

    Images

    1) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    2) Product Images from "Amide-Based Prodrugs of Spermidine-Bridged Dinuclear Platinum. Synthesis, DNA Binding, and Biological Activity"

    Article Title: Amide-Based Prodrugs of Spermidine-Bridged Dinuclear Platinum. Synthesis, DNA Binding, and Biological Activity

    Journal:

    doi: 10.1021/jm070813z

    (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by TaKaRa Taq DNA polymerase on the pSP73 plasmid DNA linearized by Hpa I restriction enzyme and subsequently modified by platinum complexes. The gel contained
    Figure Legend Snippet: (A) Autoradiogram of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by TaKaRa Taq DNA polymerase on the pSP73 plasmid DNA linearized by Hpa I restriction enzyme and subsequently modified by platinum complexes. The gel contained

    Techniques Used: Sequencing, Inhibition, DNA Synthesis, Plasmid Preparation, Modification

    3) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    4) Product Images from "Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR"

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst016

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .
    Figure Legend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Techniques Used: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.
    Figure Legend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.
    Figure Legend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation

    5) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    6) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    7) Product Images from "Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method"

    Article Title: Scarless assembly of unphosphorylated DNA fragments with a simplified DATEL method

    Journal:

    doi: 10.1080/21655979.2017.1308986

    Principle and optimization of sDATEL assembly method. (A) Schematic diagram of sDATEL. The length of overhang was set as 30 nt while the primers for amplifying DNA fragments were designed with 40 nt. After denaturation and annealing, the displaced overhangs at the fork structures were cleaved by Taq DNA polymerase and the nicks were joined by Taq DNA ligase; (B) Schematic diagram of DATEL; (C), (D) and (E) were the effects on the assembly efficiency that caused by pH, NAD+ and Mg2+ , respectively.
    Figure Legend Snippet: Principle and optimization of sDATEL assembly method. (A) Schematic diagram of sDATEL. The length of overhang was set as 30 nt while the primers for amplifying DNA fragments were designed with 40 nt. After denaturation and annealing, the displaced overhangs at the fork structures were cleaved by Taq DNA polymerase and the nicks were joined by Taq DNA ligase; (B) Schematic diagram of DATEL; (C), (D) and (E) were the effects on the assembly efficiency that caused by pH, NAD+ and Mg2+ , respectively.

    Techniques Used:

    8) Product Images from "Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR"

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst016

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .
    Figure Legend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Techniques Used: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.
    Figure Legend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.
    Figure Legend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation

    9) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    10) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    11) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    12) Product Images from "Characterization of porcine cytokine inducible SH2-containing protein gene and its association with piglet diarrhea traits"

    Article Title: Characterization of porcine cytokine inducible SH2-containing protein gene and its association with piglet diarrhea traits

    Journal: Asian-Australasian Journal of Animal Sciences

    doi: 10.5713/ajas.16.0169

    The three different Taq I PCR-RFLP genotypes of pCISH gene. The genotypes (AA, AG, GG) are shown at the top. M: DNA molecular marker DL2000. PCR-RFLP, polymerase chain reaction–restriction fragment length polymorphism; pCISH, porcine cytokine inducible SH2-containing protein.
    Figure Legend Snippet: The three different Taq I PCR-RFLP genotypes of pCISH gene. The genotypes (AA, AG, GG) are shown at the top. M: DNA molecular marker DL2000. PCR-RFLP, polymerase chain reaction–restriction fragment length polymorphism; pCISH, porcine cytokine inducible SH2-containing protein.

    Techniques Used: Polymerase Chain Reaction, Marker

    13) Product Images from "Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR"

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst016

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .
    Figure Legend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Techniques Used: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.
    Figure Legend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.
    Figure Legend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation

    14) Product Images from "Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR"

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst016

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .
    Figure Legend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Techniques Used: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.
    Figure Legend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.
    Figure Legend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation

    15) Product Images from "Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma"

    Article Title: Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma

    Journal: Journal of Asthma and Allergy

    doi: 10.2147/JAA.S37667

    Detection of cytokine messenger ribonucleic acid from lymphocytes using real-time polymerase chain reaction. Spleen cells were stimulated in vitro with OVA for 1 day in culture. Spleen cells stimulated with fetal calf serum were used as controls. Total ribonucleic acid was purified from the OVA-stimulated or fetal calf serum (control)-stimulated spleen cells using Isogen (Nippon Gene Co, Ltd, Tokyo, Japan) following the manufacturer’s instructions. For the real-time reaction, a reverse transcription system (Promega Corporation, Fitchburg, WI) was used. Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo). The specific primer pairs used were previously described. 15 The samples were amplified for 30–35 cycles under the following conditions: annealing for 30 seconds at 56°C, extension for 1 minute at 73°C, and denaturation for 30 seconds at 93°C. ( A ) The reaction products were analyzed on 2% agarose, Tris-buffered ethylenediaminetetraacetic acid gels. ( B – E ) Photographs of the gels were scanned, and band intensities were measured using a densitometer (CS Analyzer 3.0; ATTO Corporation, Tokyo, Japan). The quantity of cytokine messenger ribonucleic acid was determined by the ratio of cytokine and beta actin band intensities. Notes: * P
    Figure Legend Snippet: Detection of cytokine messenger ribonucleic acid from lymphocytes using real-time polymerase chain reaction. Spleen cells were stimulated in vitro with OVA for 1 day in culture. Spleen cells stimulated with fetal calf serum were used as controls. Total ribonucleic acid was purified from the OVA-stimulated or fetal calf serum (control)-stimulated spleen cells using Isogen (Nippon Gene Co, Ltd, Tokyo, Japan) following the manufacturer’s instructions. For the real-time reaction, a reverse transcription system (Promega Corporation, Fitchburg, WI) was used. Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo). The specific primer pairs used were previously described. 15 The samples were amplified for 30–35 cycles under the following conditions: annealing for 30 seconds at 56°C, extension for 1 minute at 73°C, and denaturation for 30 seconds at 93°C. ( A ) The reaction products were analyzed on 2% agarose, Tris-buffered ethylenediaminetetraacetic acid gels. ( B – E ) Photographs of the gels were scanned, and band intensities were measured using a densitometer (CS Analyzer 3.0; ATTO Corporation, Tokyo, Japan). The quantity of cytokine messenger ribonucleic acid was determined by the ratio of cytokine and beta actin band intensities. Notes: * P

    Techniques Used: Real-time Polymerase Chain Reaction, In Vitro, Purification, Polymerase Chain Reaction, Amplification

    16) Product Images from "Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo− ) DNA polymerase"

    Article Title: Unnatural imidazopyridopyrimidine:naphthyridine base pairs: selective incorporation and extension reaction by Deep Vent (exo− ) DNA polymerase

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp611

    Screening of DNA polymerases for single-nucleotide insertion. ( A ) The reaction for ImO N :NaN O base pair. ( B ) The reaction for ImN O :NaO N base pair. All reactions used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTG X GAACGGGTG-5′ (30mer, X = ImO N , NaN O , ImN O or NaO N ). Lanes 1 and 14 indicate the 20-mer primer; lanes 2, 8, 15 and 21 indicate the results of KF (exo − ); lanes 3, 9 16, and 22 indicate those of Taq DNA polymerase; lanes 4, 10, 17 and 23 indicate those of Tth DNA polymerase; lanes 5, 11, 18 and 24 indicate those of Vent (exo − ) DNA polymerase; lanes 6, 12, 19 and 25 indicate those of Deep Vent (exo − ) DNA polymerase; and lanes 7, 13, 20 and 26 indicate those of KOD Dash DNA polymerase.
    Figure Legend Snippet: Screening of DNA polymerases for single-nucleotide insertion. ( A ) The reaction for ImO N :NaN O base pair. ( B ) The reaction for ImN O :NaO N base pair. All reactions used the primer-template combination 5′-FITC-GTTCTGGATGGTCAGCGCAC-3′ (20mer) and 3′-CAAGACCTACCAGTCGCGTG X GAACGGGTG-5′ (30mer, X = ImO N , NaN O , ImN O or NaO N ). Lanes 1 and 14 indicate the 20-mer primer; lanes 2, 8, 15 and 21 indicate the results of KF (exo − ); lanes 3, 9 16, and 22 indicate those of Taq DNA polymerase; lanes 4, 10, 17 and 23 indicate those of Tth DNA polymerase; lanes 5, 11, 18 and 24 indicate those of Vent (exo − ) DNA polymerase; lanes 6, 12, 19 and 25 indicate those of Deep Vent (exo − ) DNA polymerase; and lanes 7, 13, 20 and 26 indicate those of KOD Dash DNA polymerase.

    Techniques Used:

    17) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    18) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    19) Product Images from "Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR"

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    doi: 10.1093/dnares/dst016

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .
    Figure Legend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Techniques Used: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.
    Figure Legend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Techniques Used: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.
    Figure Legend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Techniques Used: Generated, Polymerase Chain Reaction, Plasmid Preparation

    20) Product Images from "Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0123468

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Modification, Polymerase Chain Reaction, Mutagenesis

    Related Articles

    DNA Extraction:

    Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
    Article Snippet: Paragraph title: DNA extraction, PCR amplification, and genotyping ... Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water.

    Electrophoresis:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 20 s and extension at 72°C for 25 s (the annealing temperature used for the amplification of Mu*8 was 62.9°C).

    Amplification:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Both the wild-type and mutant templates (plasmids) were amplified using an allele-specific 3'-ddC-blocked forward primer and an unblocked reverse primer. .. The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: In the new strategy, we selected the ddNTP-blocked primer, which appears to have the best blocking effect , as the allele-specific primer to improve the specificity of PR-PCR. .. In addition, we largely decreased the amount of high-fidelity DNA polymerase to avoid the non-specific primer extension and added a routine amount of Taq DNA polymerase into the reaction to improve amplification efficiency. .. The major role of the tiny amount of high-fidelity DNA polymerase in the reaction is to activate primer extension by specifically removing the 3'-blocked nucleotide under the presence of a mismatch between the primer and the template ( ).

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Then we selected 62.2°C as the annealing temperature of the modified PR-PCR to further optimize the amount of high-fidelity DNA polymerase. .. 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 ( ). .. Thus, a mix of 0.1–0.15 U of high-fidelity DNA polymerase with 1 U of Taq DNA polymerase in per 20 μL reaction was selected for the subsequent experiments.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: With each of these two plasmids serving as template, the same primer pair from pHsh-kan described above was used to construct random mutagenesis libraries by in situ error-prone PPCP. .. The amplification was performed over celA and celB using Taq DNA polymerase in the presence of 0.1 mM Mn2+ and Tma DNA ligase. .. The amplification products were transformed into competent E. coli cells, and the mutant libraries screened on the LB plates containing kanamycin and CMC.

    Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
    Article Snippet: After preliminary screening from universal plastid and nuclear primers, four cpDNA intergenic spacers, psb A-trn H (Chiang and Peng, ), psb M-trn D (Shaw et al., ), trn S-trn G (Shaw et al., ), and trn L-trn T (Taberlet et al., ) and three nuclear genes, GTP , guanosine triphosphate (GTP) gene (Salas-Leiva et al., ); PHYP , phytochrome P gene and PPRC , hypothetical protein gene (unpublished) were chose for complete analysis (Table ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. PCR amplifications were performed in a thermocycler under the following conditions: an initial 5 min denaturation at 80°C, followed by 29 cycles of 1 min at 95°C, 1 min annealing at 50°C, and a 1.5 min extension at 65°C, and a final extension for 5 min at 65°C for cpDNA intergenic spacers.

    Agarose Gel Electrophoresis:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 20 s and extension at 72°C for 25 s (the annealing temperature used for the amplification of Mu*8 was 62.9°C).

    In Situ:

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Paragraph title: In situ error-prone PPCP mutagenesis ... Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

    Polymerase Chain Reaction:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Both the wild-type and mutant templates (plasmids) were amplified using an allele-specific 3'-ddC-blocked forward primer and an unblocked reverse primer. .. The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 20 s, annealing at 62.2°C for 20 s and extension at 72°C for 25 s (the annealing temperature used for the amplification of Mu*8 was 62.9°C).

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: PCR was performed in 50 μl reaction solution containing 1.25 U Pyrobest DNA polymerase (TaKaRa) for 30 cycles, each cycle consisting of incubation at 94°C for 40 s, 52°C for 40 s and 72°C for 1 min 10 s. The 894 bp dsDNA fragment generated was subsequently used as PPCP primer pair for amplifying whole pHsh plasmid DNA as described below. .. The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ . .. PCR was performed for 25 cycles with each cycle consisting of incubation at 94°C for 30 s (denaturation), 62°C for 30 s (annealing), 72°C for 1.5 min (extension) and 60°C for 2 min (ligation).

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Two most common rifampin-resistant mutations, H526Y ( C AC > T AC) and S531L (T C G > T T G) [ ] in the rpoB gene of TB were detected using the modified PR-PCR. .. All reactions were performed in a total volume of 20 μL containing a mixture of 2 μL of (10×) Taq PCR buffer, 0.2 mM dNTPs, 1 U of Taq DNA polymerase, 0.15 U of PrimeSTAR HS DNA polymerase, 5 ng of gDNA (equal to approximately 1×106 copies of the TB genome) and 0.3 μM each of 3'-blocked forward primer and common reverse primer. .. The PCR cycling conditions consisted of pre-denaturation at 95°C for 1 min, followed by 35 cycles of denaturation at 95°C for 15 s, annealing at 59°C for 15 s and extension at 72°C for 15 s. To confirm the results obtained from the modified PR-PCR, a 493 bp fragment of the rpoB gene encompassing the two mutation sites from six TB strains was sequenced using the 454 GS-FLX system (Roche Diagnostics Corporation).

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: For other details, please see . (B) Gel electrophoresis of the amplicons obtained using the AS-PCR with 5 ng of genomic DNA. .. All reactions were performed in a total volume of 20 μL containing a mixture of 0.5 U of Taq DNA polymerase, 0.3 μM each of forward and reverse primer, 0.2 mM dNTPs, (1×) PCR Buffer and 5 ng (equal to approximately 1×106 copies) of template. .. The PCR cycling condition was pre-denaturation at 98°C for 2 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 55°C for 15 s and extension at 72°C for 15 s. The positive (TB-526PC and TB-531PC) and negative control plasmids were both amplified, regardless of whether WT-F or Mu-F was used.

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The extracted gDNA was eluted into 70 μL of (1×) TE buffer (pH 8.0) and then quantified using a Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). .. For the clinical samples, the modified PR-PCR was performed in a total volume of 25 μL reaction mixture including 20 ng of gDNA, 3.75 μL of (5×) PrimeSTAR buffer, 1.25 μL of (5×) Taq PCR buffer, 0.2 mM dNTPs, 0.2 U of PrimeSTAR HS DNA polymerase, 0.7 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of reverse primer, fusion-blocked forward primer and adaptor. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min; 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 20 s and extension at 72°C for 18 s. The PCR products were visualized via 1.5% agarose gel electrophoresis.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: In contrast, an increasing amount of circular DNA product was obtained with increasing amount of Tma DNA ligase added to the reaction mixture (lanes 2–5, Fig. ), indicating the need to determine the sufficient amount of DNA ligase required for optimal ligation to compensate for enzyme inactivation at high temperature during the denaturation step of the cycle. .. When Taq DNA polymerase is employed under error-prone conditions, e.g. in the presence of Mn2+ (mutagenic PCR), PPCP can be used for generating random mutagenesis libraries. .. Since mutagenesis occurs in situ during the PCR reaction, eliminating the need of a separate process for generating or sub-cloning mutant DNA, we designated the procedure as in situ error-prone PPCP.

    Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
    Article Snippet: After preliminary screening from universal plastid and nuclear primers, four cpDNA intergenic spacers, psb A-trn H (Chiang and Peng, ), psb M-trn D (Shaw et al., ), trn S-trn G (Shaw et al., ), and trn L-trn T (Taberlet et al., ) and three nuclear genes, GTP , guanosine triphosphate (GTP) gene (Salas-Leiva et al., ); PHYP , phytochrome P gene and PPRC , hypothetical protein gene (unpublished) were chose for complete analysis (Table ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. PCR amplifications were performed in a thermocycler under the following conditions: an initial 5 min denaturation at 80°C, followed by 29 cycles of 1 min at 95°C, 1 min annealing at 50°C, and a 1.5 min extension at 65°C, and a final extension for 5 min at 65°C for cpDNA intergenic spacers.

    Mutagenesis:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Both the wild-type and mutant templates (plasmids) were amplified using an allele-specific 3'-ddC-blocked forward primer and an unblocked reverse primer. .. The PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Paragraph title: In situ error-prone PPCP mutagenesis ... Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: In contrast, an increasing amount of circular DNA product was obtained with increasing amount of Tma DNA ligase added to the reaction mixture (lanes 2–5, Fig. ), indicating the need to determine the sufficient amount of DNA ligase required for optimal ligation to compensate for enzyme inactivation at high temperature during the denaturation step of the cycle. .. When Taq DNA polymerase is employed under error-prone conditions, e.g. in the presence of Mn2+ (mutagenic PCR), PPCP can be used for generating random mutagenesis libraries. .. Since mutagenesis occurs in situ during the PCR reaction, eliminating the need of a separate process for generating or sub-cloning mutant DNA, we designated the procedure as in situ error-prone PPCP.

    Blocking Assay:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The ddNTP-blocked primer can not be extended by DNA polymerase because of the lack of free 3'-OH. .. We first performed AS-PCR assays with Taq DNA polymerase to test and compare the blocking efficiencies of the three kinds of inert primers mentioned above. .. A wild-type template that completely matched the allele-specific blocked primer and a mutant template that contained a C-T mismatch with the 3'-end of the allele-specific primer were used in the reactions ( ).

    Purification:

    Article Title: Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma
    Article Snippet: Total RNA was purified from OVA-stimulated or fetal calf serum (control)-stimulated spleen cells using Isogen (Nippon Gene Co, Ltd, Tokyo, Japan) following the manufacturer’s instructions. .. Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo).

    Real-time Polymerase Chain Reaction:

    Article Title: Single systemic administration of Ag85B of mycobacteria DNA inhibits allergic airway inflammation in a mouse model of asthma
    Article Snippet: Paragraph title: Detection of cytokine mRNA from lymphocytes using real-time polymerase chain reaction ... Polymerase chain reaction was performed in a total volume of 50 μL of 1 × polymerase chain reaction buffer (Takara Shuzo, Kyoto, Japan) containing 0.5–1.0 μg of complementary DNA, 0.25 mM of each deoxyribonucleotide triphosphate, 2 μM of each primer, and 2.5 U of Taq DNA polymerase (Takara Shuzo).

    Modification:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: These indicate that the modified PR-PCR can be used in long-fragment amplification as well. .. In conclusion, we developed a novel modified PR-PCR method that uses a ddNTP-blocked primer and an enzyme combination of a routine amount of Taq DNA polymerase and a tiny amount of high-fidelity DNA polymerase. .. This method can be used for easy detection of various mutation types, including point mutations, insertions and deletions, with high selectivity and sensitivity.

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The major role of the tiny amount of high-fidelity DNA polymerase in the reaction is to activate primer extension by specifically removing the 3'-blocked nucleotide under the presence of a mismatch between the primer and the template ( ). .. To determine the appropriate amount of high-fidelity DNA polymerase for the modified PR-PCR, we performed a preliminary experiment using a combination of 1 U of Taq DNA polymerase together with 0.05, 0.1 or 0.3 U of KOD FX DNA polymerase at an annealing temperature of 56°C in a 20 μL reaction ( ). .. The result showed that when the amount of high-fidelity DNA polymerase was decreased to 0.05 U, only the mutant sequence could be slightly amplified , indicating that the decrease in the amount of high-fidelity DNA polymerase indeed inhibited the non-specific amplification of the wild-type sequence and enhanced the discrimination efficiency.

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: The extracted gDNA was eluted into 70 μL of (1×) TE buffer (pH 8.0) and then quantified using a Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE). .. For the clinical samples, the modified PR-PCR was performed in a total volume of 25 μL reaction mixture including 20 ng of gDNA, 3.75 μL of (5×) PrimeSTAR buffer, 1.25 μL of (5×) Taq PCR buffer, 0.2 mM dNTPs, 0.2 U of PrimeSTAR HS DNA polymerase, 0.7 U of Taq DNA polymerase, 1 μL of DMSO and 0.3 μM each of reverse primer, fusion-blocked forward primer and adaptor. .. The reactions were carried out under cycling conditions of pre-denaturation at 94°C for 2 min; 40 cycles of denaturation at 98°C for 10 s, annealing at 59°C for 20 s and extension at 72°C for 18 s. The PCR products were visualized via 1.5% agarose gel electrophoresis.

    Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
    Article Snippet: We extracted genomic DNA from dried leaves using the modified CTAB method (Doyle, ). .. Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water.

    Selection:

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .

    Sequencing:

    Article Title: Species Delimitation of the Cycas segmentifida Complex (Cycadaceae) Resolved by Phylogenetic and Distance Analyses of Molecular Data
    Article Snippet: Amplification protocols were as follows: for cpDNA, each 30μL reaction contained 15 ng DNA, 3.0μL 10 × PCR buffer, 1.5μL MgCl2 (25mM), 1.5μL dNTPs (10mM), 1.5μL DMSO, 0.45μL of each primer, 0.45μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 19.5μL double-distilled water; for nuclear genes, the PCR reactions contained 30 ng DNA, 3.0μL 10 × PCR buffer, 2.25μL MgCl2 (25mM), 2.25μL dNTPs (10mM), 2.25 DMSO, 0.6μL of each primer, 0.5μL Taq DNA polymerase (5 U/μL) (Takara, Shiga, Japan) and 15.53μL double-distilled water. .. All PCR products of different DNA fragments were sequenced directly in both directions by the dideoxy chain-termination method, using an ABI 3730XL automated sequencer (made in Applied Biosystems) at Shanghai Meiji Biological Medicine and Technology Co Ltd.

    other:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: Thus, a mix of 0.1–0.15 U of high-fidelity DNA polymerase with 1 U of Taq DNA polymerase in per 20 μL reaction was selected for the subsequent experiments.

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: We further performed the routine PR-PCR assay using high-fidelity DNA polymerase instead of Taq DNA polymerase.

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: S1 Fig (a) The conventional AS-PCR mediated by Taq DNA polymerase. (b) The typical PR-PCR method mediated by high fidelity DNA polymerase and ddC-blocked primer.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Error-prone PPCP using Taq DNA polymerase in the presence of Mn2+ , Tma DNA ligase and the PPCP primer pair from pHsh-kan was performed over the xynA 1 region of pHsh-xynA 1 template (Fig. ).

    Plasmid Preparation:

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: PCR of 30 cycles was performed in 50 μl of reaction solution containing 1.25 U Pyrobest DNA polymerase (TaKaRa), each cycle consisting of incubation at 94°C for 30 s, 54°C for 30 s and 72°C for 2 min. .. Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ . .. Endoglucanase gene celA was amplified by PCR using the genomic DNA of T. maritima as template and the following primers: 5′-CTGTGGTACTGATGACAAAACCGGGAACATC-3′ and 5′-GGGAAGCTTTCATTCTCTCACCTCCAGATC-3′.

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR
    Article Snippet: Paragraph title: Amplification of whole pHsh plasmid DNA by PPCP with simultaneous selection maker swapping ... The PPCP experiment shown in Fig. was performed in 20 μl reaction solution containing 50 ng pHsh-Amp (2.44 kb) as template, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 80 ng PPCP primer pair prepared by PCR as described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 and 0.5 mM NAD+ .

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 97
    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: 97/100, based on 215 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerase/product/TaKaRa
    Average 97 stars, based on 215 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2019-12
    97/100 stars
      Buy from Supplier

    99
    TaKaRa la taq polymerase
    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the <t>Taq</t> DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; <t>Takara-bio).</t> PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.
    La Taq Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 490 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/la taq polymerase/product/TaKaRa
    Average 99 stars, based on 490 article reviews
    Price from $9.99 to $1999.99
    la taq polymerase - by Bioz Stars, 2019-12
    99/100 stars
      Buy from Supplier

    92
    TaKaRa qrt pcr reactions
    The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. <t>qRT-PCR</t> was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P
    Qrt Pcr Reactions, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/qrt pcr reactions/product/TaKaRa
    Average 92 stars, based on 8 article reviews
    Price from $9.99 to $1999.99
    qrt pcr reactions - by Bioz Stars, 2019-12
    92/100 stars
      Buy from Supplier

    Image Search Results


    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 PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer.

    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 PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer.

    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 PCR amplifications were performed in a total volume of 20 μL with 2 μL of (10×) Taq PCR buffer, 0.2 mM deoxynucleotide-triphosphate (dNTPs), 1 ng of template, 0.15 units (U) of KOD FX DNA polymerase (TOYOBO, Osaka, Japan), 1 U of Taq DNA polymerase (TaKaRa, Dalian, China) and 0.3 μM each of reverse primer and 3'-ddC-blocked forward primer.

    Techniques: Modification, Polymerase Chain Reaction, Mutagenesis

    Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    doi: 10.1093/dnares/dst016

    Figure Lengend Snippet: Directed evolution of xylanse and cellulase as examples of in situ error-prone mutagenic PCR mutagenesis using thermostable DNA ligase-mediated PPCP. (A) Agarose gel analysis of the PPCP mutagenesis products using pHsh- xynA 1 as template and the PPCP primer pair from pHsh- kan . Lanes: M, λ EcoT14\xE2\x85 markers; 1, control reaction containing no Taq DNA polymerase or Tma ligase; 2, control reaction containing Taq DNA polymerase but no Tma ligase; 3, PCR containing both Taq DNA polymerase and Tma ligase. (B) Functional screening of E. coli transformants for xylanase activity using xylan-overlay assay as described in Materials and methods. Positive clones were identified by clear zones surrounding xylanase-expressing colonies (arrows). The mutants were derived from pHsh- xynA 1. The size of xynA 1 was 591 bp. (C) Functional screening of E. coli transformants for cellulase activity by CMC-overlay assay as described in Materials and methods. Positive clones were identified by depressed area surrounding cellulase-expressing colonies (arrows). The mutants shown were derived from pHsh- celB .

    Article Snippet: Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

    Techniques: In Situ, Polymerase Chain Reaction, Mutagenesis, Agarose Gel Electrophoresis, Functional Assay, Activity Assay, Overlay Assay, Clone Assay, Expressing, Derivative Assay

    Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    doi: 10.1093/dnares/dst016

    Figure Lengend Snippet: Agarose gel analysis of the thermostable DNA ligase-mediated PPCP using pHsh- amp as template. Lane M, DL5000 markers; Lane 1, control reaction containing template plasmid and primer but no Taq DNA polymerase or ligase; Lane 2, control reaction containing Taq DNA polymerase but no ligase; Lane 3, PPCP reaction containing both Taq DNA polymerase and Tma DNA ligase. The upper band in lane 2 is presumed to be linear PCR product. Incorporating the nick ligation step resulted in a nick-free, circular PPCP product (lane 3). The size of the PPCP primer pair was 962 bp.

    Article Snippet: Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

    Techniques: Agarose Gel Electrophoresis, Plasmid Preparation, Polymerase Chain Reaction, Ligation

    Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Thermostable DNA Ligase-Mediated PCR Production of Circular Plasmid (PPCP) and Its Application in Directed Evolution via In situ Error-Prone PCR

    doi: 10.1093/dnares/dst016

    Figure Lengend Snippet: Optimization of thermostable DNA ligase-mediated PPCP by varying the amounts of Tma DNA ligase in 20 μl reaction. Lane M: λ EcoT14\xE2\x85markers; Lanes 1–5: 0, 0.06, 0.1, 0.2 and 0.3 μg of Tma DNA ligase, respectively. The PPCP long primer pair (2361 bp) was generated using pHshRev and pHshFwd as PCR primers, pHsh- kan as template, and Pyrobest DNA polymerase as described in Materials and methods. PPCP was carried out using pHsh- xynA 1 as template and Taq DNA polymerase as described in Materials and methods. The unlabelled, upper band is presumed to be un-ligated, open circle plasmid as its amount diminished with the increasing amount of Tma ligase.

    Article Snippet: Error-prone PPCP was performed in 20 μl reaction solution containing 30 ng plasmid pHsh-xynA 1, pHsh-celA or pHsh-celB (see below for their preparations) as template as described in the above, 0.5 U Taq DNA polymerase (TaKaRa), 0.1 μg/μl Tma DNA ligase, 200 ng PPCP primer pair from pHsh described above, 0.2 mM each deoxynucleotide triphosphate, 2.0 mM MgCl2 , 0.5 mM MnCl2 and 0.5 mM NAD+ .

    Techniques: Generated, Polymerase Chain Reaction, Plasmid Preparation

    PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: PCR of subdivided genomic sequences. Three regions of human genomic DNA (GenBank accession nos AC006454 , AC093734 and X91835 , with 5103, 5193 and 12 114 bp, respectively) were subdivided into nine 567, 577 and 1346 bp PCR sites, respectively. PCR was performed for each subdivided site using primer sets (20 bp each) corresponding to the terminal sequence of each site using the Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio). PCR was carried out in the absence and in the presence of Tth RecA protein and ATP. The products were electrophoresed and stained with ethidium bromide. ( a ) A diagrammatic representation of the subdivided region (5103 bp in GenBank accession no AC006454 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( b ) A diagrammatic representation of the subdivided region (5193 bp in GenBank accession no AC093734 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). ( c ) A diagrammatic representation of the subdivided region (12 114 bp in GenBank accession no X91835 ) (upper panel) and the electrophoretic patterns of the PCR products (lower panel). Throughout (a–c), nine subdivided sites for each region are indicated as a-1 to a-9, b-1 to b-9 and c-1 to c-9. Nucleotide (nt) numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction, Genomic Sequencing, Sequencing, Staining

    PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: PCR with primers carrying mismatched bases. PCR was performed at two human genomic sites with primers (20 bp), one of which (forward primer) carried one, two or three mismatched bases in the middle of the primer, in the absence (left) or presence (right) of Tth RecA protein and ATP using the Taq DNA polymerase ( rTaq DNA polymerase plus ‘hot start’ antibody). ( a ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 66 562 and nt 66 581 in GenBank accession no AC006454 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set a-1); lanes 2 and 6, PCR products using primers (primer set a-2 with one mismatched base at nt 66 566, T to C); lanes 3 and 7, PCR products using primers (primer set a-3 with two mismatched base at nt 66 566 and nt 66 571, both T to C); and lanes 4 and 8, PCR products using primers (primer set a-4 with three mismatched base at nt 66 566 and nt 66 571, T to C and nt 66 576, G to C). The oligonucleotide sequences used for the forward primers (mismatched bases are underlined) are as follows: primer set a-1, 5′-CATGGCACCTGCTCTGAGAC-3′; primer set a-2, 5′-CATGGCACC C GCTCTGAGAC-3′; primer set a-3, 5′-CATGGCACC C GCTC C GAGAC-3′; and primer set a-4, 5′-CATG C CACC C GCTC C GAGAC-3′. ( b ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 38 501 and nt 38 520 in GenBank accession no. AC0937734 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set b-1); lanes 2 and 6, PCR products using primers (primer set b-2 with one mismatched base at nt 38 505, G to A); lanes 3 and 7, PCR products using primers (primer set b-3 with two mismatched base at nt 38 505 and nt 38 510, both G to A); and lanes 4 and 8, PCR products using primers (primer set b-4 with three mismatched base at nt 38 505, nt 38 510 and nt 38 515, all G to A). The oligonucleotide sequences used for the forward primers are as follows: primer set b-1, 5′-ATCTGTGTGGTTCGGCTCTG-3′; primer set b-2, 5′-ATCTGTGTG A TTCGGCTCTG-3′; primer set b-3, 5′-ATCTGTGTG A TTCG A CTCTG-3′; and primer set b-4, 5′-ATCT A TGTG A TTCG A CTCTG-3′. ( c ) Upper panel: a diagrammatic representation of the location of the PCR site (20 bp between nt 63 957 and nt 63 976 in GenBank accession no. AC004975 ) and of the primers. Lower panel: lanes 1 and 5, PCR products using primers without mismatched bases (primer set c-1); lanes 2 and 6, PCR products using primers (primer set c-2 with one mismatched base at nt 63 961, A to T); lanes 3 and 7, PCR products using primers (primer set c-3 with two mismatched base at nt 63 961 and nt 63 966, A to T and C to T); and lanes 4 and 8, PCR products using primers (primer set c-4 with three mismatched base at nt 63 961, nt 63 966 and nt 63 971, A to T, C to T and G to T). The oligonucleotide sequences used for the forward primers are as follows: primer set c-1, 5′-GCAGGCACCAAGAACTACTG-3′; primer set c-2, 5′-GCAGGCACC T AGAACTACTG-3′; primer set c-3, 5′-GCAGGCACC T AGAA T TACTG-3′; and primer set c-4, 5′-GCAG T CACC T AGAA T TACTG-3′. The sequences for the backward primers are 5′-TCACCTCCCAGCCTGGCCCA-3′ for ( a ), 5′-AGGGAGATGTTCTCATAAAT-3′ and 5′-CTGTAAGTGGCAGACATTAC-3′ for ( b ). Nucleotide numbers correspond to registries in GenBank. Locations of the specific PCR products are indicated by arrows.

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction

    Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Journal: Nucleic Acids Research

    Article Title: Multiplex PCR: use of heat-stable Thermus thermophilus RecA protein to minimize non-specific PCR products

    doi: 10.1093/nar/gni111

    Figure Lengend Snippet: Effect of T.thermophilus RecA protein on PCR. PCR with Taq DNA polymerase ( ExTaq DNA polymerase plus ‘hot start’ antibody; Takara-bio) for several randomly selected sequences (300–650 bp) in human genomic DNA was carried out in the absence or in the presence of the Tth RecA protein. ( a ) Control, PCR under the standard conditions described in Materials and Methods. ( b ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture). ( c ) Similar to (a), but with ATP (400 μM). ( d ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP (300 μM). ( e ) Similar to (a), but with Tth RecA protein (0.4 μg per 25 μl reaction mixture) and ATP-γS (300 μM). The products were electrophoresed and stained with ethidium bromide. Molecular weight markers are indicated on the right-hand side of these panels. The oligonucleotide sequences used for the primers were as follows: 5′-ACAATGGGCTCACTCACCCA-3′ and 5′-CTAAGACCAATGGATAGCTG-3′ for lane 1 (300 bp); 5′-GCTCAGCATGGTGGTGGCAT-3′ and 5′-CCTCATACCTTCCCCCCCAT-3′ for lane 2 (319 bp); 5′-GACTACTCTAGCGACTGTCC-3′ and 5′-GACAGCCACCAGATCCAATC-3′ for lane 3 (360 bp); 5′-AACCTCACAACCTTGGCTGA-3′ and 5′-TTCACAACTTAAGATTTGGC-3′ for lane 4 (400 bp); 5′-AGGCAACTAGGATGGTGTGG-3′ and 5′-CAGGGAGCGTGTCCATAGGG-3′ for lane 5 (450 bp); 5′-CTGCTGAAAGAGATGCGGTG-3′ and 5′-AGGAAAACAGCCCAAGGGAC-3′ for lane 6 (469 bp); and 5′-ACTTTGTTCTGAGCCTCACA-3′ and 5′-GTTGCCCAATCGCCCCTCTC-3′ for lane 7 (650 bp).

    Article Snippet: The same results were obtained by the other polymerase systems: LA Taq polymerase (Takara-bio), Tth polymerase (Applied-boisystems); Expand High Fidelity, Expand High FidelityPLUS and Expand Long Template polymerase (Roche-diagnostics); TITANIUM Taq polymerase (Becton-Dickinson-Clontech); and Taq polymerase (Promega).

    Techniques: Polymerase Chain Reaction, Staining, Molecular Weight

    The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: The impact of TUG1 on tumorigenesis in vivo . ( a and b ) Scramble or shTUG1 was transfected into SPC-A1 cells, which were injected in the nude mice ( n =10), respectively. Tumor volumes were calculated after injection every 2 days. Bars indicate S.D. ( c ) Tumor weights are represented as means of tumor weights ±S.D. qRT-PCR was performed to detect the average expression of TUG1. ( d ) Histopathology of xenograft tumors. The tumor sections were under H E staining and IHC staining using antibodies against Ki-67. Bar, 100 μ m. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: In Vivo, Transfection, Injection, Mouse Assay, Quantitative RT-PCR, Expressing, Histopathology, Staining, Immunohistochemistry

    TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: TUG1 could epigenetically regulate HOXB7 by binding to PRC2. ( a ) qRT-PCR was performed to detect the expression of HOX genes in transfected cells, and western blot assays were used to detect the level of HOXB7 after the transfection of si-TUG1. IHC assays were used to detect the HOXB7 in tumor sections from shTUG1-transfected cells. ( b ) RIP experiments were performed in SPC-A1 and the coprecipitated RNA was subjected to qRT-PCR for TUG1. HOTAIR was used as a positive control. The fold enrichment of TUG1 in EZH2 RIP is relative to its matching IgG control RIP. TUG1 nuclear localization, as identified using qRT-PCR in fractionated SPC-A1 and A549 cells. ( c ) ChIP of H3K27me3 and EZH2 of the promoter region of HOXB7 locus after siRNA treatment targeting si-NC or si-TUG1; qPCR were performed to determine the quantitation of ChIP assays. The levels of qPCR products are expressed as a percentage of the input DNA. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Binding Assay, Quantitative RT-PCR, Expressing, Transfection, Western Blot, Immunohistochemistry, Positive Control, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Quantitation Assay

    p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: p53 induces TUG1 through interacting with the promoter region of TUG1. ( a ) Analysis of TUG1 expression levels in NSCLC cell lines (A549, SPC-A1, SK-MES-1, NCI-H1299 and NCI-H1650) compared with the normal bronchial epithelial cell line (16HBE) by qRT-PCR. ( b ) Description of p53RE and mutant p53RE in promoter region of TUG1. The position of ChIP primers ( e ) was indicated by arrows. ( c ) Western blotting was used to detect the p53 induction by doxo. qRT-PCR was used to detect the effect of doxo on TUG1 expression in p53-WT and p53-null cells. ( d ) Induction of TUG1 by ectopically expressed p53 (wild-type p53 or mutant p53). Overexpression was confirmed by western blotting. ( e ) Induction of TUG1 promoter activity by p53, but not mutant p53 in NCI-H1299 cell lines. ( f ) Deletion and mutation analysis of the promoter activity to determine the role of the p53RE in p53-mediated regulation of TUG1. ( g ) The p53 binding at the promoter regions of TUG1 was assessed by ChIP analysis. ChIP primers were detailed in Materials and Methods section. Shown are representative images of three independent experiments. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR, Mutagenesis, Chromatin Immunoprecipitation, Western Blot, Over Expression, Activity Assay, Binding Assay

    Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: Analysis of TUG1 expression in NSCLC tissues and clinical parameters. ( a ) TUG1 was detected in 192 pairs of NSCLC tissues by qRT-PCR. The levels of TUG1 in NSCLC tissues are significantly lower than those in non-tumorous tissues. The ΔCt value was determined by subtracting the GAPDH Ct value from the TUG1 Ct value (relative to a single reference value). Smaller ΔCt value indicates higher expression. ( b and c ) Data are presented as relative expression level in tumor tissues (shown as ΔCt). TUG1 expression was significantly lower in patients with a higher pathological stage and big tumor size. ( d ) Patients with low levels of TUG1 expression showed reduced survival times compared with patients with high levels of TUG1 expression ( P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: Expressing, Quantitative RT-PCR

    TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

    Journal: Cell Death & Disease

    Article Title: P53-regulated long non-coding RNA TUG1 affects cell proliferation in human non-small cell lung cancer, partly through epigenetically regulating HOXB7 expression

    doi: 10.1038/cddis.2014.201

    Figure Lengend Snippet: TUG1 could participate in AKT and MAPK pathway through modulating HOXB7. ( A ) MTT analysis of cell proliferation by co-transfection (si-NC, si-HOXB7, si-HOXB7+si-TUG1). At 48 h after transfection of si-HOXB7, the cell cycle and apoptosis were analyzed by flow cytometry. LR, early apoptotic cells. UR, terminal apoptotic cells. ( B ) qRT-PCR were performed to detect the expression of HOXB7 after overexpression of p53 and transfected with p53 followed by transfection with si-TUG1. ( C ) Western blotting analysis of the expression of p-ERK, total ERK, p-AKT, total AKT, p-GSK-3 β , total GSK-3 β proteins in indicated si-NC-transfected, si-HOXB7 and si-TUG1-transfected SPC-A1 cell lines. ( D ) Immunostaining of HOXB7 was negatively or very weakly positive in corresponding non-tumor lung tissues (a and b), but was strongly positive in squamous cell carcinoma tissues (c and d) and lung adenocarcinoma (e and f). Bar, 100 μ m. ( E ) The immunoreactivity of HOXB7 protein in NSCLC tissues showed a statistically significant inverse correlation with the relative level of TUG1 expression. Error bars indicate means±S.E.M. * P

    Article Snippet: After the RT reaction, 1 μ l of the complementary DNA was used for subsequent qRT-PCR reactions (SYBR Premix Ex Taq, TaKaRa) according to the manufacturer's instructions.

    Techniques: MTT Assay, Cotransfection, Transfection, Flow Cytometry, Cytometry, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Immunostaining