摘要
目的:利用长距离PCR定点突变技术对GEFS+致病相关基因SCN1A进行定点诱变。方法:在突变位点处设计一对延伸方向相反的引物,通过长距离PCR扩增,得到携带突变位点的新质粒;然后用Dpn I酶切去掉母质粒;再转化至大肠杆菌进行克隆。结果:DNA测序表明:在预期位点已经发生突变,SCN1A所编码的第1765位密码子由苯丙氨酸(Phe)突变为亮氨酸(Leu)。结论:SCN1A突变表达载体的构建成功,为进一步研究该突变位点导致I型电压依赖型钠通道功能的改变奠定了基础。
Objective: Long-distance PCR was applied to site-directed mutagenesis of epilepsy-related gene SCN1A in vitro. Methods: A pair of primers was designed at the mutation site and mutational plasmids were obtained by long-distance PCR. And then Dpn I was used to digest parental plasmids. Digested PCR product was transformed into E. coli competent cells to amplify mutational plasmids. Results: As shown by DNA sequencing, mutation was identified at the directed site as SCNIA F1765U Conclusion: the successfully constructed mutational plasmids SCN1A F1765L may provide a molecular basis for further functional and genomic investigation of SCN1A.
出处
《广州医学院学报》
2007年第2期62-64,共3页
Academic Journal of Guangzhou Medical College
基金
国家自然科学基金(30600198)
广东省自然科学基金博士科研启动项目(06301101)
广东省名医工程研究项目(2004-18)
关键词
聚合酶链反应
诱变
定点
SCN1A
癫痫
polymerase chain reaction
mutagenesis,site-directed
SCN1A
epilepsy