一种移码差错率大幅度下降的聚合酶N端缺失体的研究

A Sludy of a Derivative of Taq DNA Polymerase

采用双引物定点突变法，构建了以２３６位氨基酸为起始密码子的Ｔａｑ酶（ＴａｑＮＤ２３６）的高表达质粒。并以－１移码突变质粒ｐＦＤＰＭＩ１８作为模板，建立了测定ＤＮＡ聚合酶的体外合成精确性的Ｇａｐｐｅｄ－ＤＮＡ系统。通过转化大肠杆菌ＴＧ１菌株后计数Ｘ－ｇａｌ平板上蓝白菌落的比例，测定了Ｔａｑ和ＴａｑＮＤ２３６两种ＤＮＡ聚合酶在ＤＮＡ体外合成中的移码突变率，发现缺失后的ＴａｑＮＤ２３６复制精确性提高了１０倍以上。

sing the method of double primer oligonucleotide-mediated mutagenesis,thehigh expression plasmid of TaqND236,a derivative of Taq DNA polymerase,wasconstructed.To determine the frameshift mutation frequency of the in vitro DNAsynthesis,we constructed a Gapped-DNA system using the pFDPM118 (a mutant ofpUC118 with a-1 frameshift mutation on the lacZ gene)as template,By calculatingthe ratio of blue and white colonies on the X-gal plate after transforming E.coliTG1 the frameshift mutation frequency of Taq and TaqND236 was measured,It was found that the replication fidelity of the deleted Taq -TaqND236 increased morethan 10 folds.