taq dna polymerase  (TaKaRa)

 
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    TaKaRa Taq DNA Polymerase
    Description:
    TaKaRa Taq DNA Polymerase is a recombinant version Taq polymerase derived from the Thermus aquaticus YT 1 strain and is suitable for routine PCR applications
    Catalog Number:
    r001c
    Price:
    None
    Size:
    3 000 Units
    Category:
    Takara Taq and premix Takara Taq products Standard PCR PCR
    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.
    TaKaRa Taq DNA Polymerase is a recombinant version Taq polymerase derived from the Thermus aquaticus YT 1 strain and is suitable for routine PCR applications
    https://www.bioz.com/result/taq dna polymerase/product/TaKaRa
    Average 99 stars, based on 2134 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerase - by Bioz Stars, 2020-08
    99/100 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 "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:

    3) Product Images from "Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain"

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain

    Journal: Neuroscience letters

    doi: 10.1016/j.neulet.2010.12.060

    (A) Genotype in each generation of mutant mice confirmed by PCR analysis on isolated genomic DNA. PCR was performed on the isolated genomic DNA using taq DNA polymerase and primer sets (#1 and #2 are for wild-type and GluR2 delta7 KI, #3 and #4 are for
    Figure Legend Snippet: (A) Genotype in each generation of mutant mice confirmed by PCR analysis on isolated genomic DNA. PCR was performed on the isolated genomic DNA using taq DNA polymerase and primer sets (#1 and #2 are for wild-type and GluR2 delta7 KI, #3 and #4 are for

    Techniques Used: Mutagenesis, Mouse Assay, Polymerase Chain Reaction, Isolation

    4) 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

    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 "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

    8) 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

    9) Product Images from "Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease"

    Article Title: Improvement of long segment ribosomal PCR amplification for molecular identification of Litylenchus crenatae mccannii associated with beech leaf disease

    Journal: Journal of Nematology

    doi: 10.21307/jofnem-2020-016

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with Dream Taq ™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Fall specimens with Dream Taq ™. M: DNA markers; 1: 104H78; 2: 104H81; 3: 104H82; 4: 104H83; 5: 104H84; 6: 104H85; 7: 104H86; 8: 104H87; 9: 104H88; 10: 104H89; 11: 104H90; NC: negative control. 1-7: Female; 8-11: Male.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of TaKaRa Ex Taq ® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq ® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq ® system and Dream Taq ™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.
    Figure Legend Snippet: PCR performance of TaKaRa Ex Taq ® system and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1 and 5: 104K37; 2 and 6: 104K38; 3 and 7: 104K39; 4 and 8: 104K40. A: 1, 2, 3 and 4: TaKaRa Ex Taq ® system; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System; B: 1, 2, 3 and 4: TaKaRa Ex Taq ® system and Dream Taq ™; 5, 6, 7 and 8: PicoMaxx™ High Fidelity PCR System. NC: negative control, respectively.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: Dream Taq ™; 5, 6, 7 and 8: Pfu ; 9, 10, 11and 12: Dream Taq ™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ or/and Pfu in PicoMaxx™ buffer. M: DNA markers; 1, 2, 3 and 4: Dream Taq ™; 5, 6, 7 and 8: Pfu ; 9, 10, 11and 12: Dream Taq ™ and Pfu combined; 1, 5 and 9: 104K29; 2, 6 and 10: 104K30; 3, 7 and 11: 104K31; 4, 8 and 12: negative control (NC), respectively.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both Dream Taq ™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: Dream Taq ™ PCR buffer; B: Pfu PCR buffer.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with both Dream Taq ™ and Pfu in manufacturer’s PCR buffers. M: DNA markers; 1: 104K29; 2: 104K30; 3: 104K31; NC: negative control, respectively. A: Dream Taq ™ PCR buffer; B: Pfu PCR buffer.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq ® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: Dream Taq ™; B: 18 S locus (1.7 kb) by Dream Taq ™, C: ITS and 28 S loci (1.9 kb) by Dream Taq ™; D: TaKaRa Ex Taq ® system.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with TaKaRa Ex Taq ® system. M: DNA markers; 1: 104J54; 2: 104J55; 3: 104J58; 4: 104J59; NC: negative control, respectively. A: Dream Taq ™; B: 18 S locus (1.7 kb) by Dream Taq ™, C: ITS and 28 S loci (1.9 kb) by Dream Taq ™; D: TaKaRa Ex Taq ® system.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: Dream Taq ™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: Dream Taq ™ and Pfu ; 4, 5 and 6: Dream Taq ™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and Dream Taq ™ in 0.625 units.
    Figure Legend Snippet: PCR performance of Pfu and Pwo in PicoMaxx™ buffer. M: DNA markers; 1 and 4: 104K37; 2 and 5: 104K38; 3 and 6: 104K39. A: 1, 2 and 3: Dream Taq ™; 4, 5 and 6: Pwo (0.125 μl per reaction). B: 1, 2 and 3: Dream Taq ™ and Pfu ; 4, 5 and 6: Dream Taq ™ and Pwo (0.125 μl per reaction). NC: negative control, respectively. Note: final concentration of Pfu in each reaction was aligned with Pwo and Dream Taq ™ in 0.625 units.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Concentration Assay

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by Dream Taq ™, B: ITS and 28 S loci (1.9 kb) by Dream Taq ™; C: Dream Taq ™ and PicoMaxx™ High Fidelity PCR System combined.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with Dream Taq ™ and PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: 18 S locus (1.7 kb) by Dream Taq ™, B: ITS and 28 S loci (1.9 kb) by Dream Taq ™; C: Dream Taq ™ and PicoMaxx™ High Fidelity PCR System combined.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    PCR performance of Taq 2000™, Platinum™ Taq and Dream Taq ™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq 2000™; 4, 5, 6 and NC by Platinum™ Taq ; 7, 8, 9 and NC by Dream Taq ™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq 2000™ or Dream Taq ™ in each reaction was aligned with Platinum™ Taq in 1.25 units.
    Figure Legend Snippet: PCR performance of Taq 2000™, Platinum™ Taq and Dream Taq ™. M: DNA markers; 1, 4 and 7: 104N95; 2, 5 and 8: 104N96; 3, 6 and 9: 104N97. 1, 2, 3 and NC by Taq 2000™; 4, 5, 6 and NC by Platinum™ Taq ; 7, 8, 9 and NC by Dream Taq ™, NC: negative control, respectively. A: 3.5 kb target; B: 1.9 kb ITS and 28 S target. Note: final concentration of either Taq 2000™ or Dream Taq ™ in each reaction was aligned with Platinum™ Taq in 1.25 units.

    Techniques Used: Polymerase Chain Reaction, Negative Control, Concentration Assay

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K17; 2: 104K18; 3: 104K19; 4: 104K20; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.
    Figure Legend Snippet: Long range ribosomal PCR Amplifications of the 3.5 kb target from Summer specimens with PicoMaxx™ High Fidelity PCR System. M: DNA markers; 1: 104K25; 2: 104K26; 3: 104K27; 4: 104K28; 5: 104K29; 6: 104K30; 7: 104K31; NC: negative control, respectively. A: Dream Taq ™; B: PicoMaxx™ High Fidelity PCR System.

    Techniques Used: Polymerase Chain Reaction, Negative Control

    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 "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

    13) 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

    14) 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

    15) 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

    16) 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

    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 "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

    20) Product Images from "An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents"

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0020303

    HotStart and low-DNA Taq DNA polymerases are not sufficiently pure for sensitive and specific broad-range amplification of bacterial DNA. The genomic DNA (100 fg) of S. aureus was amplified by HotStart or low-DNA Taq DNA polymerases (Taq #1: Hot Start Taq DNA polymerase, Protech Inc.; Taq #2: Fast Hot Start Taq DNA polymerase, KAPA Biosystems; Taq #3: Taq DNA polymerase, TakaRa Inc.; Taq #4: ULTRATOOLS Taq DNA polymerase, Biotools Inc.) using the primer set p201 and p1370 (lanes 1, 3, 5, and 7). Significant amount of PCR product was present in the no template control reactions (lanes 2, 4, 6, and 8).
    Figure Legend Snippet: HotStart and low-DNA Taq DNA polymerases are not sufficiently pure for sensitive and specific broad-range amplification of bacterial DNA. The genomic DNA (100 fg) of S. aureus was amplified by HotStart or low-DNA Taq DNA polymerases (Taq #1: Hot Start Taq DNA polymerase, Protech Inc.; Taq #2: Fast Hot Start Taq DNA polymerase, KAPA Biosystems; Taq #3: Taq DNA polymerase, TakaRa Inc.; Taq #4: ULTRATOOLS Taq DNA polymerase, Biotools Inc.) using the primer set p201 and p1370 (lanes 1, 3, 5, and 7). Significant amount of PCR product was present in the no template control reactions (lanes 2, 4, 6, and 8).

    Techniques Used: Amplification, Polymerase Chain Reaction

    21) 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

    22) 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

    23) 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

    24) 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

    25) 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

    26) 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

    Related Articles

    Clone Assay:

    Article Title: Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium
    Article Snippet: .. The thermal cycling protocol consisted of an initial denaturation step at 95°C for 1 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min. Adenine was added to the 3′ terminus of the PCR product by using Taq polymerase (Takara Shuzo), and the product was cloned into pT7Blue by TA cloning. .. A unique Asc I site within sthA in the PCR product was used as the insertion site of the Kmr gene cassette as follows.

    Amplification:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: .. The results showed that the use of 0.1–0.15 U of high-fidelity DNA polymerase combined with 1 U of Taq DNA polymerase in per 20 μL reaction resulted in the best discrimination and amplification effects ( ). .. 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.

    Mutagenesis:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: .. Development of a modified PR-PCR method using the dideoxynucleotide-blocked primer and a mixture of Taq DNA polymerase and high-fidelity DNA polymerase The experimental results above indicate that two major factors affect the efficiency of the PR-PCR in mutation detection. .. One is that the modification of the 3'-end of a primer with-NH2 or-Pi is unable to completely block primer extension ( ).

    Isolation:

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain
    Article Snippet: .. PCR was performed on the isolated genomic DNA using taq DNA polymerase (TaKaRa Ex Taq™, Takara, Otsu, Japan) and primer sets (primer #21–23 for GluR2 delta7: #21, 5′-ACA GAG GAA GGT AGT GGA AGG GAG-3′; #22, 5′-CTT GGT TTG GTT GTT GGT CAT AGC-3′; #23, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′; primer #31–33 for GluR3 delta7: #31, 5′-CCA ATA CTC CAC AGG GGC AAT TTA TC-3′; #32, 5′-CCG TTG ACT GTT TTG AAT CTC ACA CC-3′; #33, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′). .. Size of the amplification product was estimated by gel electrophoresis for genotyping (210 bp for wild-type and 350 bp for GluR2 delta7; 440 bp for wild-type and 300 bp for GluR3 delta7).

    TA Cloning:

    Article Title: Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium
    Article Snippet: .. The thermal cycling protocol consisted of an initial denaturation step at 95°C for 1 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min. Adenine was added to the 3′ terminus of the PCR product by using Taq polymerase (Takara Shuzo), and the product was cloned into pT7Blue by TA cloning. .. A unique Asc I site within sthA in the PCR product was used as the insertion site of the Kmr gene cassette as follows.

    Polymerase Chain Reaction:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: .. 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: Identification of a Gene Essential for Sheathed Structure Formation in Sphaerotilus natans, a Filamentous Sheathed Bacterium
    Article Snippet: .. The thermal cycling protocol consisted of an initial denaturation step at 95°C for 1 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 2 min. Adenine was added to the 3′ terminus of the PCR product by using Taq polymerase (Takara Shuzo), and the product was cloned into pT7Blue by TA cloning. .. A unique Asc I site within sthA in the PCR product was used as the insertion site of the Kmr gene cassette as follows.

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain
    Article Snippet: .. PCR was performed on the isolated genomic DNA using taq DNA polymerase (TaKaRa Ex Taq™, Takara, Otsu, Japan) and primer sets (primer #21–23 for GluR2 delta7: #21, 5′-ACA GAG GAA GGT AGT GGA AGG GAG-3′; #22, 5′-CTT GGT TTG GTT GTT GGT CAT AGC-3′; #23, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′; primer #31–33 for GluR3 delta7: #31, 5′-CCA ATA CTC CAC AGG GGC AAT TTA TC-3′; #32, 5′-CCG TTG ACT GTT TTG AAT CTC ACA CC-3′; #33, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′). .. Size of the amplification product was estimated by gel electrophoresis for genotyping (210 bp for wild-type and 350 bp for GluR2 delta7; 440 bp for wild-type and 300 bp for GluR3 delta7).

    other:

    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. ).

    Modification:

    Article Title: Modified Proofreading PCR for Detection of Point Mutations, Insertions and Deletions Using a ddNTP-Blocked Primer
    Article Snippet: .. Development of a modified PR-PCR method using the dideoxynucleotide-blocked primer and a mixture of Taq DNA polymerase and high-fidelity DNA polymerase The experimental results above indicate that two major factors affect the efficiency of the PR-PCR in mutation detection. .. One is that the modification of the 3'-end of a primer with-NH2 or-Pi is unable to completely block primer extension ( ).

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

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain
    Article Snippet: .. PCR was performed on the isolated genomic DNA using taq DNA polymerase (TaKaRa Ex Taq™, Takara, Otsu, Japan) and primer sets (primer #21–23 for GluR2 delta7: #21, 5′-ACA GAG GAA GGT AGT GGA AGG GAG-3′; #22, 5′-CTT GGT TTG GTT GTT GGT CAT AGC-3′; #23, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′; primer #31–33 for GluR3 delta7: #31, 5′-CCA ATA CTC CAC AGG GGC AAT TTA TC-3′; #32, 5′-CCG TTG ACT GTT TTG AAT CTC ACA CC-3′; #33, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′). .. Size of the amplification product was estimated by gel electrophoresis for genotyping (210 bp for wild-type and 350 bp for GluR2 delta7; 440 bp for wild-type and 300 bp for GluR3 delta7).

    Activated Clotting Time Assay:

    Article Title: Involvement of GluR2 and GluR3 subunit C-termini in the trigeminal spinal subnucleus caudalis and C1-C2 neurons in trigeminal neuropathic pain
    Article Snippet: .. PCR was performed on the isolated genomic DNA using taq DNA polymerase (TaKaRa Ex Taq™, Takara, Otsu, Japan) and primer sets (primer #21–23 for GluR2 delta7: #21, 5′-ACA GAG GAA GGT AGT GGA AGG GAG-3′; #22, 5′-CTT GGT TTG GTT GTT GGT CAT AGC-3′; #23, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′; primer #31–33 for GluR3 delta7: #31, 5′-CCA ATA CTC CAC AGG GGC AAT TTA TC-3′; #32, 5′-CCG TTG ACT GTT TTG AAT CTC ACA CC-3′; #33, 5′-CTA GTG AAC CTC TTC GAG GGA C-3′). .. Size of the amplification product was estimated by gel electrophoresis for genotyping (210 bp for wild-type and 350 bp for GluR2 delta7; 440 bp for wild-type and 300 bp for GluR3 delta7).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    TaKaRa race pcr kit
    siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by <t>RACE.</t> Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental <t>RT-PCR</t> (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p
    Race Pcr Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/race pcr kit/product/TaKaRa
    Average 99 stars, based on 6 article reviews
    Price from $9.99 to $1999.99
    race pcr kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    99
    TaKaRa pcr mimic construction kit
    DEX effects on RA synovial fibroblast annexin I mRNA. Panel (a): RNA extracted from human RA FLS was subjected to competitive <t>RT-PCR</t> with (lanes 2–6 and 8–12) or without (lanes 1 and 7) <t>mimic.</t> Lanes 1–6: control; lanes 7–12: DEX 10 −7 M for 24 hours. Annexin I mRNA content was greater in DEX-treated than vehicle-treated cells. Representative of n = 4 separate experiments from 4 separate RA donors. Panel (b): the log of the ratios of the annexin I and mimic PCR product band intensities were graphed as a function of the log of the amount of mimic added to the reaction. Increased annexin I mRNA is seen in DEX-treated RA synovial fibroblasts. Representative of n = 4 separate experiments from 4 separate RA donors.
    Pcr Mimic Construction Kit, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pcr mimic construction kit/product/TaKaRa
    Average 99 stars, based on 3131 article reviews
    Price from $9.99 to $1999.99
    pcr mimic construction kit - by Bioz Stars, 2020-08
    99/100 stars
      Buy from Supplier

    Image Search Results


    siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p

    Journal: PLoS Pathogens

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms

    doi: 10.1371/journal.ppat.1007534

    Figure Lengend Snippet: siRNA (-2752-21) targets tomato SlLNR1 . (A) Schematic illustration of SlLNR1 structure and its associated siRNAs. The sense SlLNR1 , SlLNR1 (+), from the susceptible cultivar is an 1132-nt lncRNA, which is validated by RACE. Its anti-sense, SlLNR1 (-), is generated by sequencing a segmental RT-PCR (the 54 th bp of the 3’ end to the 1008 th ). The full sequence of SlLNR1 was shown in S4 Fig . The vertical bar indicate the target site of siRNA(-2752-21). The data derived from small RNA sequencing of TYLCV infected plants or mock was aligned to SlLNR1 and the number indicates the aligned siRNA reads. (B) Negative correlation of expression of siRNA(-2752-21) and SlLNR1 . SlLNR1 (+) or SlLNR1 (-) was expressed with siRNA(-2752-21) in N . benthamiana . The RNA sample was extracted at 48 h after agroinfiltration. The EF1a gene of N . benthamiana was used as a refernce. (C) SlLNR1 was down regulated in the pTRV2:IR inoculated susceptible plants but not in the resistant plants. The RNA sample was extracted at 15 days after pTRV2:IR and EV inoculated plants. The relative expression of SlLNR1 was measured by qRT-PCR and calculated in relation to EV inoculated plants according to the ΔΔ Ct method. The tomato actin gene was set as reference gene. Error bars represented SE of three biological replicates and significant differences by Student’s t test (*, p

    Article Snippet: The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA).

    Techniques: Generated, Sequencing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, RNA Sequencing Assay, Infection, Expressing, Quantitative RT-PCR

    Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p

    Journal: PLoS Pathogens

    Article Title: Tomato yellow leaf curl virus intergenic siRNAs target a host long noncoding RNA to modulate disease symptoms

    doi: 10.1371/journal.ppat.1007534

    Figure Lengend Snippet: Identification of a 25-nt segment and a vsRNA that induce stunt and curled leaves in tomato. (A) VsRNAs generated by the IR and sequence alignment of vsRNAs and SlLNR1 . Location and frequency of TYCLV-derived siRNAs (vsRNAs) were mapped to the IR in sense- (above the x-axis) or antisense- (below the x-axis) orientation. Genome organization of the IR was shown at the top in which the inverted repeat was symbolized as a stem loop. Numbers indicate the first (2616) and last (147) nucleotides of the IR sequence. The histogram of location, frequency and size distribution of vsRNAs corresponding to the 25-nt-fragment (2730–2754) were shown at the medium panel. The fragment was highly complemented with SlLNR1 (-). The scissor means the cleavage site determined by 5’-RACE analysis. (B) Phenotypes of tomato inoculated with pTRV2:4TR. TYLCV-susceptible tomato plants were inoculated with pTRV2 containing 4×25-nt-fragment (2730–2754). The photos were taken at 15 dpi. (C) Validation of siRNA(-2752-21) in the tomato plants by siRNA Northern blot. The leaves of susceptible tomato plants inoculated by TYLCV infectious clone, natural infection by viruliferous whiteflies, agroinfiltrated with pTRV2:4TR and pTRV2:IR were used for total RNA extraction and analyzed at 24 dpi. U6 gene was set as the internal control. (D) Validation of siRNA(-2752-21) presence and downregulation of SlLNR1 in the overexpressed plants. Two individual transgenic lines (pCAMBIA2301:siRNA-1/2) with overexpression of siRNA(-2752-21) were used for total RNA extraction and analyzed. EV indicates the transgenic plant with the EV. The lower panel shows the relative expressi o n of SlLNR1 that was measured by qRT-PCR and calculated in relation to the transgenic plants according to the ΔΔ Ct method using tomat o actin gene as the reference. Error bars represented SE of three biological replicates and significant differences by Student’ s t test (*, p

    Article Snippet: The 5’ flanking region of the sense transcripts of SlLNR1 were obtained by RNA ligase-mediated rapid amplification of 5' cDNA ends First Choice ® RLM-RACE Kit (Invitrogen, USA), according to the instructions of the manufacturer, and the 3’ end was verified by 3’ RACE PCR kit (TAKARA).

    Techniques: Generated, Sequencing, Derivative Assay, Northern Blot, Infection, RNA Extraction, Transgenic Assay, Over Expression, Quantitative RT-PCR

    DEX effects on RA synovial fibroblast annexin I mRNA. Panel (a): RNA extracted from human RA FLS was subjected to competitive RT-PCR with (lanes 2–6 and 8–12) or without (lanes 1 and 7) mimic. Lanes 1–6: control; lanes 7–12: DEX 10 −7 M for 24 hours. Annexin I mRNA content was greater in DEX-treated than vehicle-treated cells. Representative of n = 4 separate experiments from 4 separate RA donors. Panel (b): the log of the ratios of the annexin I and mimic PCR product band intensities were graphed as a function of the log of the amount of mimic added to the reaction. Increased annexin I mRNA is seen in DEX-treated RA synovial fibroblasts. Representative of n = 4 separate experiments from 4 separate RA donors.

    Journal: Mediators of Inflammation

    Article Title: Regulation of Annexin I in Rheumatoid Synovial Cells by Glucocorticoids and Interleukin-1

    doi: 10.1155/MI/2006/73835

    Figure Lengend Snippet: DEX effects on RA synovial fibroblast annexin I mRNA. Panel (a): RNA extracted from human RA FLS was subjected to competitive RT-PCR with (lanes 2–6 and 8–12) or without (lanes 1 and 7) mimic. Lanes 1–6: control; lanes 7–12: DEX 10 −7 M for 24 hours. Annexin I mRNA content was greater in DEX-treated than vehicle-treated cells. Representative of n = 4 separate experiments from 4 separate RA donors. Panel (b): the log of the ratios of the annexin I and mimic PCR product band intensities were graphed as a function of the log of the amount of mimic added to the reaction. Increased annexin I mRNA is seen in DEX-treated RA synovial fibroblasts. Representative of n = 4 separate experiments from 4 separate RA donors.

    Article Snippet: Briefly, PCR MIMICs were generated by two successive PCR amplifications according to the PCR MIMIC construction kit (Clontech Laboratories, Inc, Palo Alto, Calif).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Tissue distribution of human GPAT4. Quantitative PCR analysis was performed as described under “Experimental Procedures” using human normal tissue cDNA panel obtained from BioChain ( A ) or PrimGen ( B ). Data were expressed as mean ±

    Journal:

    Article Title: AGPAT6 Is a Novel Microsomal Glycerol-3-phosphate Acyltransferase *

    doi: 10.1074/jbc.M708151200

    Figure Lengend Snippet: Tissue distribution of human GPAT4. Quantitative PCR analysis was performed as described under “Experimental Procedures” using human normal tissue cDNA panel obtained from BioChain ( A ) or PrimGen ( B ). Data were expressed as mean ±

    Article Snippet: The human GPAT3 was obtained similarly by PCR amplification of a human Heart Marathon Ready cDNA library (Clontech) followed by restriction digestion with HindIII and BamHI sites and subcloning into the mammalian expression vector p3xFLAG-CMV-14.

    Techniques: Real-time Polymerase Chain Reaction

    Schematic overview of the MTA-seq workflow used in this study. Primer pairs (443) were designed to generate amplicons harboring one known SNP. Highly multiplexed PCR was conducted with the TaKaRa multiplex PCR amplification kit version 2. Amplicons were end-repaired, adapter-ligated, and nick-repaired with the SPARK DNA sample prep kit for Ion Torrent. To identify individual samples, the Ion Xpress Barcode Adapters 1-96 kit was adopted to add index adapters. The barcoded library was sequenced on an Ion Proton sequencer. SNPs were called by using the “mpileup2cns” command of the VarScan software.

    Journal: Frontiers in Plant Science

    Article Title: Multiplex PCR Targeted Amplicon Sequencing (MTA-Seq): Simple, Flexible, and Versatile SNP Genotyping by Highly Multiplexed PCR Amplicon Sequencing

    doi: 10.3389/fpls.2018.00201

    Figure Lengend Snippet: Schematic overview of the MTA-seq workflow used in this study. Primer pairs (443) were designed to generate amplicons harboring one known SNP. Highly multiplexed PCR was conducted with the TaKaRa multiplex PCR amplification kit version 2. Amplicons were end-repaired, adapter-ligated, and nick-repaired with the SPARK DNA sample prep kit for Ion Torrent. To identify individual samples, the Ion Xpress Barcode Adapters 1-96 kit was adopted to add index adapters. The barcoded library was sequenced on an Ion Proton sequencer. SNPs were called by using the “mpileup2cns” command of the VarScan software.

    Article Snippet: Multiplex PCR was performed with the Multiplex PCR assay kit version 2 (TaKaRa, Kusatsu, Japan) according to manufacturer instruction, with modifications.

    Techniques: Polymerase Chain Reaction, Multiplex Assay, Amplification, Sample Prep, Software