GEFS^+致病相关基因SCN1A表达载体的定点诱变

Site-Directed Mutagenesis of Epilepsy-related Gene SCN1A in vitro

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.