论文部分内容阅读
在大肠杆菌中建立了一套用于测定ＤＮＡ聚合酶在ＰＣＲ过程中复制精确性的系统，并测定了耐热ＦＤＤＮＡ聚合酶在ＰＣＲ扩增过程中的复制精确性。这一系统主要包括以质粒ｐＵＣ１１８和ｐＵＣ１１９为出发质粒所构建的一套共６个突变质粒，分别为ｐＦＤＦＰ１１８和ｐＦＤＦＰ１１９（＋１移码突变）、ｐＦＤＦＭ１１８和ｐＦＤＦＭ１１９（－１移码突变）、ｐＦＤＦＵ１１８和ｐＦＤＦＵ１１９（碱基置换突变）。这些突变质粒均不能进行ｌａｃＺ－α互补反应，因此在含有Ｘ－Ｇａｌ和ＩＰＴＧ的培养基上菌落呈白色。同时还构建了ＰＣＲ产物克隆载体ｐＦＤＦＬ１１８和ｐＦＤＦＬ１１９。以上述突变质粒为模板进行ＰＣＲ反应，取反应产物和ｐＦＤＦＬ１１８或ｐＦＤＦＬ１１９连接后转化到大肠杆菌中。若在ＰＣＲ过程中发生回复突变，则转化子在含有Ｘ－Ｇａｌ和ＩＰＴＧ的培养基上呈蓝色。由转化子中蓝白菌落个数即可计算出ＤＮＡ聚合酶的复制差错率。用这一系统测得ＦＤＤＮＡ耐热聚合酶的复制差错率为１０－５～１０－６。
A set of systems for determining the accuracy of DNA polymerase in PCR was established in E. coli and the replication accuracy of the thermostable FD DNA polymerase during PCR amplification was determined. This system mainly includes a set of 6 mutated plasmids constructed from plasmid pUC118 and pUC119 as pFDFP118 and pFDFP119 (+1 frameshift mutation), pFDFM118 and pFDFM119 (-1 frameshift mutation), pFDFU118 and pFDFU119 (Base substitution mutation). Neither of these mutant plasmids was able to perform lacZ-alpha complementation, so colonies were white on media containing X-Gal and IPTG. PCR product cloning vectors pFDFL118 and pFDFL119 were also constructed. A PCR reaction was carried out using the above-mentioned mutant plasmid as a template, and the reaction product was transformed into E. coli after being ligated with pFDFL118 or pFDFL119. If a back-mutation occurs during the PCR, the transformant is blue on medium containing X-Gal and IPTG. According to the number of blue-white colonies in the transformants, the replication error rate of DNA polymerase can be calculated. Using this system, the error rate of replication of FD DNA thermosensitive polymerase was 10-5 ~ 10-6.