利用Pyrobest DNA聚合酶一步法进行大片段基因的定点突变  

One-Step and Cost-Effective Site-Directed Mutagenesis of Large Plasmids Using Pyrobest DNA Polymerase

作　　者：朱大兴[1] 周清华[1] 王艳萍[2] 车国卫[2] 唐小军 朱文[2] 陈小禾[2] 孙芝琳[2] 

机构地区：[1]天津医科大学总医院肺部肿瘤外科 [2]四川大学华西医院四川省肺癌分子生物学重点实验室,四川成都610041

出　　处：《生物技术通讯》2007年第4期590-593,共4页Letters in Biotechnology

基　　金：国家自然科学基金重点项目(30430300);教育部高等学校博士学科点专项科研基金(20040610060)

摘　　要：目的:介绍一种简单、快速、高效和经济的进行大片段基因定点突变的方法。方法:小量抽提含有NM23H1-EGFP融合基因的逆转录病毒真核表达质粒pLXSN-NM23H1-EGFP,体外合成突变引物对,利用高保真Pyrobest DNA聚合酶对质粒DNA进行PCR突变反应,然后用DpnⅠ限制性内切酶消化PCR产物以去除模板DNA,取适量消化产物转化大肠杆菌XL1-Blue,随机挑选克隆进行测序筛选、鉴定所需突变株。结果:在NM23H1-EGFP基因中引入了S44A、P96S、H118F、S120G、P96S-S120G等5个替换突变位点及9bp处的插入突变。结论:该方法简单、快速、高效、经济,不须纯化中间产物,不须亚克隆,突变效率几乎为100%,是一种值得推广应用的方法。Objective： A simple, rapid, efficient and cost-effective method for site-directed mutagenesis of large plasmids in a single-PCR procedure was described. Methods： This protocol was a modification of the QuikChange^○R site-directed mutagenesis kit method by using the high fidelity pyrobest DNA polymerase. Pure plasmid containing fusion gene of NM23H1 and EGFP was mini-prepared. Five pairs of mutagenic primers were synthesized in vitro and the desired five mutations, $44A, P96S, HllSF, S120G, P96S-S120G and one insertion were introduced into NM23H1-EGFP gene by PCR. After that, Dpn I was used to digest the PCR products to remove the methylated, nonmutated parental DNA template. Optimal amount of the digested products were used to transform competent E.coli XL1-Blue cells and colonies resistant to ampicillin were picked up randomly followed by being sequenced to directly identify the needed mutants. Results： The five mutations, $44A, P96S, Hll8F, S120G, P96S-S120G and one insertion were successfully introduced into NM23H1-EGFP gene, which were proved by sequencing. Conclusion： This mutagenesis approach can be performed in one tube, and does not require purification of intermediate products. No subcloning is needed. The mutagenesis efficiency of this method is almost 100%. One-step site-directed mutagenesis of large plasmids using pyrobest DNA polymerase is a simple, rapid, efficient and cost-effective method.

关 键 词：Pyrobest DNA聚合酶 定点突变 PCR 

分 类 号：Q78[生物学—分子生物学]