改进重叠延伸法引入DNA定点突变的新方法  

A New Method of Site-directed Mutagenesis by Modifying Overlap Extension PCR

作　　者：郭志义[1,2] 郝小惠[2] 延晋雷[3] 尚利梅[3] 裴鑫[2] 武慧婧[3] 赵偲宇[3] 张璇[3] 杨方[2] 

机构地区：[1]华北煤炭医学院河北省煤矿卫生与安全重点实验室 [2]华北煤炭医学院实验研究中心,河北唐山063000 [3]华北煤炭医学院生物学系

出　　处：《生物技术》2009年第6期34-36,共3页Biotechnology

摘　　要：目的:改进重叠延伸PCR法,实现一种引入DNA定点突变的准确简便方法。方法:通过应用不同的扩增酶和反应体系,以重叠延伸PCR的方法产生引入突变位点的DNA片断,然后再亚克隆到载体中。该文以人cyclin D1启动子的NF-κB位点(-39/-30)为例。结果:通过DNA测序证明定点突变成功引入。一次引入4个突变碱基。突变引入率为100%。Objective：To develop a new method for generation of site-directed mutation by modifying overlap extension PCR.Method： mutation generation by overlap extension PCR using different DNA polymerase and reaction buffer.The mutation fragment is subcloned into T-vector.In this study,we selected the NF-κB site（-39/-30）of human cyclin D1 promoter for example.Result： The mutation is verified by DNA sequencing.Four point-mutations were intruduced by one-step.The overall rate of obtaining the multiple mutant sites was 100%.Conclusion： An effective and cheap method of DNA mutation by modifying overlap extension PCR had developed.

关 键 词：定点突变 重叠延伸PCR 高GC含量 

分 类 号：Q784[生物学—分子生物学]