【摘要】 目的 :建立发掘未知单核苷酸多态性 (singlenucleotidepolymorphisms,SNPs)和已知SNPs分型的技术平台。方法 :运用PCR产物双向大规模测序的方法发掘未知SNPs;运用基于聚合酶链反应 -限制性片段长度多态性 (poly merasechainreaction restrictionendonucleasedigestion ,PCR RFLP)、TaqMan技术对已知SNPs进行分型。结果 :建立了基于PCR产物双向大规模测序发掘未知SNP的技术平台 ,并以此在 2 7个个体的 6 9个乙型肝炎候选易感基因区域检测到 5 92个SNPs,核苷酸变异度为 (4 .5 1± 1.2 4 )× 10 - 4;建成基于PCR RFLP和TaqMan的SNP分型技术平台 ,这两种方法与直接测序法比较 ,PCR RFLP的检出率达到 10 0 % ,错误率几乎为 0 ,并且操作简单 ,成本低廉 ;TaqMan分型技术的检出率也可达到 10 0 % ,与测序结果的一致性达到 10 0 % ,并且过程简单、易于操作 ,结果直观 ,易于判断 ,也能快速得到结果。结论 :基于PCR产物双向大规模测序发掘未知SNPs的技术平台成熟可靠 ,适于准确、大规模地发掘未知SNPs;PCR RFLP技术和TaqMan分型技术均适用于今后大规模正常人群和疾病人群的SNPs分型 ,为进行关联分析以确定疾病相关的SNPs奠定了坚实的技术基础。更多还原

【Abstract】 Objective:To establish the technique systems of discovering the unknown single nucleotide polymorphisms(SNPs)and genotyping the known SNPs. Methods:Large-scale sequencing of PCR products was used to discover the unknown SNPs, and PCR-RFLP and TaqMan were used to genotype the known SNPs. Results:The technique system of discovering the unknown SNPs based on large-scale sequencing of PCR products was established successfully, with which 592 SNPs in genomic regions of 69 susceptibe candidate genes for chronic hepatitis B in 27 individuals were discovered. The nucleotide diversity was (4.51 ± 1.24) × 10 -4. The genotyping technique systems including PCR-RFLP and TaqMan were also established successfully. Compared with the methods of direct sequencing, the above two kinds of genotyping methods were simple, cheap, fast, and had similar success rate. In addition, the concordance between them was 100%. Conclusions:The technique systems of discovering and genotyping SNPs are reliable. They are all suitable for large-scale discovering and genotyping SNPs, which are good tools for identifying susceptibe genes of complicated diseases.更多还原