环状定点诱变技术在单碱基定点诱变中的应用  

Site-directed mutagenesis of single base in vitro using circular site-directed mutagenesis technology

作　　者：周亚芹[1] 张洪梅[1] 董艳[1] 苏青[1] 

机构地区：[1]上海交通大学医学院附属新华医院内分泌代谢科,上海200092

出　　处：《现代医学》2009年第5期321-324,共4页Modern Medical Journal

摘　　要：目的采用环状定点诱变技术将重组真核表达质粒pcDNA3.1-hTSHR中hTSHR cDNA序列上发生突变的单碱基诱变回正常的序列。方法设计1对含预期突变的完全互补的诱变引物,以环状质粒为模板进行PCR扩增,得到带缺口的突变质粒,酶消化去除模板质粒,将PCR产物转化DH5α感受态细胞进行缺口修复和克隆。结果DNA测序表明,在预期位点已成功发生诱变,且目的基因其他部位的碱基序列均未发生随机突变。结论环状定点诱变技术是一种高效、简便、快捷的诱变方法,通过该方法成功实现了目的碱基的定点诱变,为后续研究奠定了基础。Objective To mutate single base of hTSHR cDNA subcloned into pcDNA3.1 to normal base sequence by circular site-directed mutagenesis technology. Methods A pair of completely complementary primers with desired mutation was designed, circular plasmid was used as the template, the nicked mutational plasmids were obtained by PCR amplifying. Parental plasmids were digested by specific enzyme. Digested PCR products were transformed into E. coli competent cells for repairing nick and amplifying. Results As shown by DNA sequencing, desired mutation was successfully completed, and other bases of target gene were not mutated. Conclusion Circular site-directed mutagenesis technology is an efficient and rapid method of site-directed mutagenesis. Target base was successfully mutated by this method,which provides a basis for further study.

关 键 词：环状定点突变 聚合酶链反应 质粒 人促甲状腺激素受体 

分 类 号：Q754[生物学—分子生物学]