摘要
目的构建含β地中海贫血(简称β地贫)IVS-2-654(C>T)突变基因的质粒。方法以含β珠蛋白野生型基因的质粒DNA为模板,采用重叠延伸PCR(OE-PCR)定点诱变后行TA克隆的方法构建含IVS-2-654(C>T)突变基因的质粒。结果双向测序结果表明:重组质粒的确含β地贫IVS-2-654(C>T)突变基因,β珠蛋白IVS-2-654处的碱基已由C突变成T,其余序列与野生型完全相同,成功实现了定点诱变。结论成功地构建了含β地贫IVS-2-654(C>T)突变基因的质粒,进一步为该病基因诊断与筛查技术的研究奠定了实验基础;OE-PCR定点诱变法简便、经济,值得推广应用。
Objective To construct a plasmid containing IVS-2-654（C〉T） single site mutation of β-thalassemia gene.Methods The plasmid DNA containing wild-type β-globin gene was used as PCR template.Plasmid containing IVS-2-654（C〉T） mutation gene of β-globin was constructed by TA clone technology after site-directed mutagenesis of overlap extension PCR（OE-PCR）.Results Direct DNA sequencing showed that the recombinant plasmid contained IVS-2-654（C〉T） mutation gene of β-globin,which mutated from C to T at IVS-2-654 bp and the rest was completely identical with wide type.Conclusions The plasmid containing IVS-2-654（C〉T） mutation gene was successfully constructed,which laied the foundation for further screening studies to detect gene mutation of β-thalassemia and other genetic diagnosis for this disease.Site-directed mutagenesisis of OE-PCR technique is simple and economic and worth of being promoted widely.
出处
《中国预防医学杂志》
CAS
2012年第10期747-750,共4页
Chinese Preventive Medicine
基金
温州市科技计划资助项目(Y20100036)
浙江省医药卫生科技计划资助项目(2011RCA030)
浙江省公益性技术应用研究计划资助项目(2011C37042)
