BRAF和TP53基因热点突变的构建

Construction of Hot- spot Mutations in Human BRAF and TP53 Genes

目的:构建BRAF和TP53基因多个位点的点突变质粒,为进一步研究其在肿瘤形成中的作用奠定基础。方法:以HEK-293T基因组DNA为模板构建含BRAF600、TP53175和TP53248位点的野生型质粒,利用MutanBESTTM定点突变方法获得含BRAFV600E、TP53R175C和TP53R248W等突变位点的突变型质粒,并进行DNA测序鉴定。结果:DNA测序结果显示构建的三个点突变与实验设计完全一致。结论:成功构建了BRAFV600E、TP53R175C和TP53R248W三个具有不同突变位点的突变型质粒,MutanBESTTM定点突变技术是一种简单、快速、高效的基因定点突变方法。

Objective ：The aim of this study is to construct multiple site -directed mutagenesis of BRAF and TP53 genes, and then to pro- vide basement for the studying of their function during tumor formation. Method ：The wild -type plasmids including the sites of BRAF600, TP53175 or TP5324Swere constructed after amplifying their gene fragments from HEK -293T genome. Subsequently, the corresponding mu- tant plasmids including BRAFV600E, TP53 R175c or TP53 R248W were obtained according to the procedures of the MutanBESTTM Kit, a reverse - PCR - based site - directed mutagenesis method. Then those mutant plasmids were sequenced. Result： The plasmids containing site - mu- tants of BRAFv600E, TP53R175Cor TP53R248W were sequenced. The results of DNA sequencing confirmed that the DNA sequences of mutant genes were completely concordant with experimental design. Conclusion： Three site- mutations including BRAFv600E, TP53a175c and TP53r248w were successfully constructed, and MutanBESTTM site- directed mutagenesis is a simple ,fast and efficient method.