PCR技术对Wilson病基因进行定点突变的研究  

PCR Site-Directed Mutagenesis of Wilson Disease Gene in Vitro

作　　者：闫振文[1] 梁秀龄[1] 杨春水[1] 侯国庆[1] 王莹[1] Toshihiro Sugiyama 

机构地区：[1]中山医科大学附属第一医院神经内科,广东广州510080

出　　处：《中山医科大学学报》2002年第2期94-96,F002,共4页Academic Journal of Sun Yat-sen University of Medical Sciences

基　　金：卫生部临床学科重大基金资助项目 ( 370 91) ;"2 11工程"重点建设基金资助项目;广东省自然科学基金资助项目 ( 2 0 0 10 70 5 ) ;广东省科委重点攻关基金资助项目 ( 982 7830 )

摘　　要：【目的】利用聚合酶链反应 (PCR)技术对Wilson病 (WD)基因进行体外定点突变的研究。【方法】采用PCR定点突变技术 ,首先设计两对引物 ,将突变位点设计在引物上 ,通过重叠延伸法两次PCR扩增 ,扩增片段上含有所需要的突变位点 ,最后将扩增片段克隆入pRc/CMV载体中。【结果】DNA测序表明在预期位点已经发生突变 ,WD基因第 778位密码子由精氨酸 (Arg)残基突变为亮氨酸残基 (Leu) ,用PCR定点突变技术成功构建Wilson病基因突变体。【结论】PCR技术诱导定点突变准确、高效。Wilson病基因突变体的构建成功 ,为进一步研究该突变位点导致Wilson病的发病机制和Wilson病蛋白的结构和功能的关系奠定了基础。Objective To study PCR site-directed mutagenesis of Wilson disease (WD) gene in vitro. Methods The site-directed mutagenesis of WD gene was made by using PCR. Two sets of primers were designed according to the gene sequence of WD, and mismatches were introduced into primers. Mutagenesis was performed in a two-step PCR. The amplified fragments from the second PCR which contained the mutation site were subcloned into the pRc/CMV vector. Results The sequencing analysis showed that the mutation site was correct. Mutation from Arg to Leu in 778 codon of WD gene was found. The mutation of WD gene was constructed successfully. Conclusion PCR site-directed mutagenesis method is accurate and highly efficient, and the mutant WD gene is a promising candidate for further studies.

关 键 词：肝豆状核变性 遗传学 聚合酶链反应 定点诱变 基因突变 研究 

分 类 号：R742.4[医药卫生—神经病学与精神病学]