DNA修复基因ERCC2rs50871遗传多态性的实时荧光定量PCR检测

Detection of Polymorphism of DNA Repair Gene ERCC2 rs50871 by Real-Time PCR

目的应用ABI7300实时荧光定量PCR-单核苷酸多态(SNP)等位基因分型技术建立准确、快速、经济、适应大样本检测SNP等位基因分型的改良方法。方法采用缩小反应的体系(由推荐的25μlPCR反应体系降至为10μl),降低探针的浓度和自动软件SNP分型方法,检测我国东北地区321例汉族无亲缘关系健康个体DNA样品的DNA修复基因ERCC2 rs50871遗传多态性。结果经改良方法检测获得了理想的等位基因分型,与DNA测序结果一致。ERCC2rs50871T等位基因频率为0.749,G等位基因频率为0.251,基因型分布符合Hardy-Weinberg平衡定律(χ2=0.003,df=1,P=0.96)。该位点等位基因频率分布与NCBI dbSNP数据库中中国北京汉族(HCB)群体的分布相似(P=0.06);但与HapMap-CEU(CEPH样品:祖先为北西欧人的犹他州居民)及HapMap-YRI(尼日利亚伊巴丹的约鲁巴人)的分布差异有统计学意义(均P<0.001)。结论本实验建立的改良方法经济实用,适合人类群体遗传学及流行病学大样本调查。

Objective To establish optimized （accurate, rapid, economical and large-samples） method for the detection of the allele genotypes for single nucleotide polymorphism by real-time PCR ABI 7300. Methods Total reaction system and probe concentration in present method were reduced by compared with traditional method. DNA samples of 321 Han subjects were from northeastern China. DNA repair genes ERCC2 rs50871 were genotyped. Results Optimized genotypes were obtained. The genotyping results were identical with ones of DNA sequencing. Allele frequencies were 0.251 （G）and 0.749 （T）, respectively. Distribution of genotypes were in Hardy-Weinberg equilibrium（Х^2=0.003, df=1, P=0.96）. The present study results were compared with ones of the previously reported in NCBI dbSNP populations. Frequency distribution of ERCC2 rs50871 among Han Chinese from northeastern China was similar to that of CHB population （P=0.40）; but it was in disagreement with HapMap-CEU＇ s （Utah residents with Northern and Western European ancestry from the CEPH collection） and HapMap-YRI＇s （Yornba in Ibadan, Nigeria） （All P〈0.001 ）. Conclusion This improved method is optimized and economical. It can compatibly be used in human population genetics and epidemiology study with larger sample size.