重叠延伸PCR法构建β地中海贫血基因突变质粒标准品  

Construction of Standard plasmids containing gene mutations of β-thalassemia by Overlap extension PCR

作　　者：余玲玲[1] 冯晶晶 田可港[3] 王慧燕[1] 郑晓群[1] 

机构地区：[1]温州医学院附属第二医院检验科,浙江温州325027 [2]浙江省杭州市滨江医院病理科,杭州311100 [3]温州医学院检验与生命科学学院,浙江温州325035

出　　处：《中国卫生检验杂志》2012年第10期2350-2353,共4页Chinese Journal of Health Laboratory Technology

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)

摘　　要：目的:构建含β地中海贫血(简称β地贫)-28(A>G)与IVS-2-654(C>T)突变基因的质粒标准品。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(Overlap extension PCR,OE-PCR)定点诱变后行TA克隆的方法分别构建含-28(A>G)与IVS-2-654(C>T)突变基因的质粒。结果:DNA测序表明:重组质粒1的确含β地贫-28(A>G)突变基因,β珠蛋白-28处的碱基已由A突变成G,其余序列与野生型完全相同;重组质粒2的确含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。成功实现了定点诱变。结论:分别成功地构建了含β地贫-28(A>G)与IVS-2-654(C>T)突变基因的质粒标准品,进一步为HRM技术检测β地贫基因突变方法的建立及该病其它基因诊断与筛查技术的研究奠定了实验基础。Objective：To construct standard plasmids containing -28（A 〉 G） and IVS -2 -654（C 〉 T） muta- tions gene of β - thalasscmia. Methods： The plasmids DNA containing wild - typeβ - globin gene were used as PCR templates, plasmids containing -28 （A 〉 G） and IVS -2 -654（ C 〉 T） mutations gene ofβ- globin respec- tive were constructed by TA clone technology after site - directed mutagenesis of overlap extension PCR （ OE - PCR）. Results： Direct DNA sequencing showed that the recombinant plasmid 1 really contained -28 （A 〉 G） mu- tation gene of β- globin, which mutated from A to G at - 28 bp, the rest was completely identical with wide type. And the recombinant plasmid 2 really contained IVS - 2 - 654 （ C 〉 T） mutation gene of β - globin, which mutated from C to T at IVS -2 -654 bp, the rest was completely identical with wide type. Conclusion： The plasmids con- taining - 28 （ A 〉 G） or IVS - 2 - 654（ C 〉 T） mutation gene respective, were successfully constructed, which lay the foundation for further HRM technology to detect gene mutation of β - thalassemia and other genetic diagnosis and screening techniques for this disease.

关 键 词：Β地中海贫血 基因突变 重叠延伸PCR 定点诱变 

分 类 号：R556.61[医药卫生—血液循环系统疾病]