BRAF和TP53基因热点突变的构建  

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

作　　者：张环宇[1,2] 王改平[1] 李晓芳[1] 姚谨[2] 陈倩倩[2] 樊路娟 

机构地区：[1]河南师范大学生命科学学院,河南新乡453007 [2]中国科学院上海巴斯德研究所病原诊断中心,上海200001

出　　处：《生物技术》2014年第4期55-59,共5页Biotechnology

基　　金：国家自然科学基金项目("蛋白OPN对大鼠肝再生中肝细胞增殖的作用研究";No.81200317);河南师范大学青年科学基金项目("桥蛋白(OPN)对大鼠原代肝细胞的作用研究";2013qk12)资助

摘　　要：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.

关 键 词：定点突变 BRAF TP53 反向PCR 

分 类 号：Q754[生物学—分子生物学]