摘要
近年来国外的研究表明:AIDS相关的CCR5和CCR5启动子调控元件等基因的突变直接影响着AIDS的发病过程,因此从事CCR5等基因的研究已成为HIV-1感染的热点课题。为了检测中国人群中AIDS相关基因的突变,首先需要提取外周血中单个核细胞(PBMC)的基因组DNA,而提取的基因组DNA的质量直接影响到AIDS相关基因的检测结果。以前文献报道多采用常规法分离淋巴细胞。
Objective To compare the efficiency of phenol/chloroform DNA extraction and Qiagen-kit DNA extraction and to analyze the AIDS-associated CCR5 gene mutation in Chinese population. Methods We purified the genomic DNAs of freshly isolated PBMC from 1219 Chinese by using two kinds of DNA-purification methods. One is conventional Phenol/Chloroform extraction of DNA, the other is the QIAamp Blood Kit extraction (QIAGEN, from Germany). After the genomic DNA purifications, We identified the CCR5 gene △32 mutations and its promoter regulation region mutions in these DNA samples by using the PCR amplification, Southern hybridization and direct DNA sequencing. Results The results showed that by using the conventional DNA extraction, the amount of 507 purified genomic DNA samples was unstable and variable; 5 ml of the fresh peripheral blood per person was needed At least, the purified process was time-consuming. In contrast, by using the QIAamp Blood Kit, only 200ml of fresh peripheral blood was used each time ant enough amount of genomie DNAs was purified for the mutation analyses of CCR5 gene △32 and its promoter regulation region. The genomic DNA amour and quality of 712 purified samples were stable (A260/280 ratio was over 1.75 for 100% samples by using the UV spectrophotometer). Conclusion Taken together, our data suggests that using QIAamp Blood Kit extraction has more advantages than using the conventional DNA extraction for purification of a large amount of genomic DNAs from the blood samples as well as for mutation analyses of purified genomic DNA samples.
出处
《传染病信息》
1999年第4期177-178,共2页
Infectious Disease Information
