体外定点突变PCR法构建abl T315I突变的重组质粒标准品  

Construction of plasmid standard preparations for abl T315I mutation detection by PCR-based site-directed mutagenesis in vitro

作　　者：石淙[1] 张长林[1] 简正伟[1] 江梅[1] 闻芳[1] 万腊根[1] 

机构地区：[1]南昌大学第一附属医院检验科,江西南昌330006

出　　处：《实验与检验医学》2013年第2期111-114,共4页Experimental and Laboratory Medicine

基　　金：江西省自然科学基金(201221AB205025)

摘　　要：目的利用聚合酶链反应(PCR)定点突变技术构建含人类abl基因第6号外显子片段的野生型及含T315I突变型重组质粒,作为检测abl T315I基因突变的阳性对照和阴性对照标准品。方法先设计包括突变位点的两对引物,以健康人外周血基因组DNA为模板,扩增获得野生型和突变型abl基因第6号外显子片段,将其插入pSG5M-flag载体质粒中,并将所获重组质粒分别进行酶切与测序鉴定,通过紫外分光光度计检测标本的浓度和纯度。结果DNA测序表明在预期位点上发生突变,abl基因第315位氨基酸密码子由苏氨酸(Thr)残基突变为异亮氨酸(Ile)残基,所构建abl基因野生型和突变型质粒,经酶切和测序鉴定与目的片段完全一致。结论PCR技术诱导定点突变准确、高效。所构建含野生型和T315I突变的abl基因重组质粒,可为检测abl基因T315I突变提供阴性和阳性对照以及质控品,同时也为T315I突变的相关研究奠定了基础。Objective To construct the recombinant plasmids containing the 6th exon fragment of wild-type abl and T31.5I mutant abl gene as the negative and positive control to detect T315I mutation in abl gene using the site-directed mutagenesis PCR. Methods Two pairs of primers were designed according to the gene sequence of abl, and mismatches were introduced into primers. The genomic DNA of healthy human peripheral blood serves as template. The wild type exon 6 fragment and the T315I mutant of abl were amplified by PCR. Then the PCR products were inserted into pSGSM-Flag plasmid and the recombinant plasmids were i- dentified by enzyme digestion and DNA sequencing. The purity and concentration of plasmids were estimated by ultraviolet spec- trophotometer. Results The sequencing analysis showed that the mutation site was correct. The 315th amino acid codon of abl gene mutated to Ile from Thr, indicating successful construction of mutant abl gene. Conclusion The data suggest that the PCR site-directed method has high accuracy and efficiency. Recombinant plasmids containing wild type exon 6 and the T315I mutant of abl were constructed successfully, which could serve as the negative and positive controls when we detect the T315I mutation of abl gene in further study.

关 键 词：定点突变 abl基因 T315I 阳性对照 

分 类 号：R733.72[医药卫生—肿瘤] Q343.13[医药卫生—临床医学]