摘要
目的 检测中国人基因组中HIV-1感染相关的特异性协同受体(CCR5)的基因多态性和比较提取人基因组DNA方法的优缺点。方法 我们分别用常规法和柱层析法从1219名中国人外周血PBMC中提取基因组DNA样品,对纯化后的DNA样品质量进行定性和定量分析;并对其基因组DNA中CCR5基因进行了PCR、Southern杂交和直接DNA测序等分析。结果 在中国人基因组中,检测的CCR5基因多态性是wt/wt 1 210例、wt/△32 3例,未检测到CCR5△32/△32纯合子突变,CCR5等位基因突变率为0.125％。常规法提取的基因组DNA所需的外周静脉血多(5ml);DNA的量较少且不稳定;操作步骤复杂。相反,QIAamp Boold Kit柱层析法提取DNA仅需要外周血200μl,可稳定地获得一定量的DNA,操作简便。结论 首次报道了中国人CCR5等位基因突变率是0.125％,提示我国绝大多数人群对嗜巨细胞HIV-1病毒的遗传易感性较高;同时证实试剂盒法提取的DNA更适合于CCR5基因多态性分析。
To compare the efficiency of phenol/chloroform DNA extraction and Qiagen - kit DNA extraction for analyzing the AIDS associated CCR5 gene mutation in Chinese population. Methods The genomic DNA of freshly isolated PBMC from 1 219 Chinese individuals were pruified by using the conventional Phenol/Choloroform and the QIAamp Blood Kit extraction, respectively. The CCR5 gene polymorphism were identified by employing the PCR amplification, Southern hybridization and direct DNA sequencing. Results Of 1 219 individuals examined, 1 210 ones had the wt/wt genotype, three with wt/△32 genotype and no one with the △32/△ 32 homozygote. Also it was found that the conventionally purified 507 samples were unstable and variable in the guantity of genomic DNAs by using 5ml of the fresh peripheral blood each time at the biginning, and the pruified process was time-consuming. In contrast, for the QIAamp Blood Kit extraction, enough amount of genomic DNAs for the analysis of CCR5 gene polymorphism could be obtained only from 200μl of fresh peripheral blood each time, and the quantity and quality of 712 DNA purified by it were stable. Conclusion Taken together, the data suggest that Chinese individuals have a significantly lower frequency of CCR5A32 mutation(0.125% ) and a high susceptibility to macrophage tropic HIV - 1 infection. Using the Kit extraction of DNA samples for the analysis of CCR5 gene polymorphism has more advantages than using the conventional DNA extraction.
出处
《中国性病艾滋病防治》
2000年第1期5-8,共4页
Chinese Journal of Std & Aids Prevention and Control
基金
国家自然科学基金资助项目(课题号:39770683)
