摘要
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。
system used for the determination of fidelity of DNA polymerase in PCR was developed in Ecoli and was used to determine the fidelity of FD DNA polymerase in PCR amplicationFrame shift and base substitution mutations were created in vitro in the lacZ gene in pUC118 and pUC119As a result,a set of six derived plasmids namely pFDFM118 and pFDFM119 (-1 frame shift),pFDFP118 and pFDFP119 (+1 frame shift),pFDFU118 and pFDFU119 (base substitution)were obtainedAll of them failed to carry out lacZ α-complementation in Ecoli MV1184 and the colonies appeared white on medium with X-Gal and IPTG consequentlyPCR reaction was carried out using these derived plasmids as templates and the PCR products were ligated to specially constructed cloning vectors pFDFL118 or pFDFL119, and the ligated products were used to transform MV1184If any back mutation happens to occur during PCR,the transformants would appear blue on medium with X-Gal and IPTGBy scoring the number of blue and white colonies,the fidelity of DNA polymerase can be calculatedWith this system the error of replication of the FD DNA polymerase was found to be 10-5～10-6
基金
国家"863"高科技发展计划生物技术领域资助
关键词
聚合酶链反应
聚合酶
复制精确性
移码突变
PCR,DNA polymerase,Fidelity of replication,Base substitution mutation,Frame shift mutation
