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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) mutation in β-thalassemia major. Methods Plasmid DNA containing the wild-type β-globin gene was used as template to amplify the plasmid containing IVS-2-654 (C> T) by TA cloning using OE-PCR. Results The two-way sequencing results showed that the recombinant plasmid did contain the gene of βS thalassemia intermedia (IVS-2-654), the base of β-globin IVS-2-654 had been mutated from C to T, Exactly the same type, successfully implemented a site-specific mutagenesis. Conclusion The plasmid containing the βS thalassemia intervertebralis IVS-2-654 (C> T) gene was successfully constructed, which laid the foundation for further research on the gene diagnosis and screening of the disease. The OE-PCR site- Economic, it is worth promoting application.