基于重叠延伸PCR法的定点突变技术  

Site-directed Mutagenesis Based on Overlap Extension PCR

作　　者：戴灿[1] 苗聪秀[1] 卢光琇[1] 

机构地区：[1]中南大学生殖与干细胞工程研究所,人类干细胞国家工程研究中心,湖南长沙410078

出　　处：《现代生物医学进展》2010年第3期411-412,共2页Progress in Modern Biomedicine

基　　金：国家高技术研究发展计划(863项目)(2006AA02A102);国家重点基础研究发展计划(973项目)(2007CB948103)

摘　　要：目的:建立一种高效而经济的定点突变方法。方法:采用重叠延伸PCR定点突变技术,引物设计时引入目的突变,以前两次PCR产物为模板,进行第三次PCR,即可获得突变后的目的DNA片段。将此片段连入pMDTM18-T载体后测序验证突变结果。结果:DNA测序表明,待突变位点已由ATTGG突变为ATTTT。结论:成功实现了目的位点的定点突变,重叠延伸PCR法是一种高效且经济的定点突变方法。Objective：To establish a fast, saving method for site-directed mutagenesis. Methods：Overlap extension PCR was used. Briefly, target mutation was introduced into primers, and the two previous PCR products were used as template for the third PCR. The final PCR segment with target mutant was then cloned into pMD？ 18-T vector for sequencing. Results：DNA sequencing showed that the target site ATTGG had been changed into ATTTT. Conclusion：Site-directed mutagenesis was successfully implemented based on the overlap extension PCR which is a fast and saving method.

关 键 词：重叠延伸PCR 定点突变 

分 类 号：Q75[生物学—分子生物学] Q78