基于重叠延伸PCR法的定点突变技术.pdfVIP

现代生物医学进展 Progress in Modern Biomedicine Vol.10 NO.03 JAN.2010 ·411 · 基于重叠延伸PCR 法的定点突变技术* 戴 灿 苗聪秀 卢光琇△ （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） : : PCR 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 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 :DNA ATTGG ATTTT : PCR 果 测序表明，待突变位点已由 突变为 。结论成功实现了目的位点的定点突变，重叠延伸 法是一种高 效且经济的定点突变方法。 关键词：重叠延伸PCR ；定点突变 Q75,Q78,R392 A 1673-6273(2010)03-411-02 中图分类号： 文献标识码： 文章编号： Site-directed Mutagenesis Based on Overlap Extension PCR* DAI Can, MIAO Cong -xiu, LU Guang -xiu△ (Institute of Reproductive and Stem Cell Engineering, Central South University, National Engineering and Research Center of Human Stem Cell, Changsha, 4 10078, China) ABSTRACT Obj ective: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. Key words: Overlap extension PCR; Site-directed mutagenesis Chinese Library Classification: Q75, 78, R392 Document code: Article ID: 1673-6273(2010)03-4 11-02 Ta