Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.
Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and vent DNA polymerases.
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DOI：
10.1101/gr.1.1.63
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发表时间：
1991-08
期刊：
影响因子：
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通讯作者：
L. Ling;P. Keohavong;C. Dias;W. Thilly
中科院分区：
文献类型：
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作者：
L. Ling;P. Keohavong;C. Dias;W. Thilly
聚合酶链式反应(PCR)中DNA聚合酶的保真度受反应混合物中多种因素的影响。为了最大限度地提高DNA聚合酶在PCR中的保真度，改变了pH、脱氧核苷三磷酸和镁离子的浓度。用变性梯度凝胶电泳法从野生型DNA序列中分离出聚合酶诱导的突变体。对不耐高温的T7 DNA聚合酶、耐热的Taq和Vent DNA聚合酶进行了研究。三种DNA聚合酶的保真度均对脱氧核苷三磷酸、镁离子和pH敏感。在允许有效扩增的条件下，关于这三个因素的优化得到了错误/碱基对的平均错误率：Taq为7.2×10(-5)，Vent为4.5×10(-5)，修饰的T7(Sequenase)DNA聚合酶为4.4×10(-5)。
The fidelity of DNA polymerases used in the polymerase chain reaction (PCR) can be influenced by many factors in the reaction mixture. To maximize the fidelity of DNA polymerases in the PCR, pH, concentrations of deoxynucleoside triphosphates, and magnesium ion were varied. Denaturing gradient gel electrophoresis was used to separate the polymerase-induced mutants from wild-type DNA sequences. Thermolabile modified T7 DNA polymerase, thermostable Taq, and Vent DNA polymerases were studied. Fidelity of all three DNA polymerases was sensitive to concentrations of deoxynucleoside triphosphates, magnesium ion, and pH. Within conditions that permitted efficient amplification, optimization with regard to these three factors yielded an average error rate in error/base pair incorporated of 7.2 x 10(-5) for Taq, 4.5 x 10(-5) for Vent, and 4.4 x 10(-5) for modified T7 (Sequenase) DNA polymerases.