基于PCR和位点特异性引物延伸反应的SNP检测方法的建立

摘要

目的 建立一种基于聚合酶链反应(PCR)、位点特异性引物延伸反应(ASE)和核酸试纸条检测技术的单核苷酸多态性(SNP)检测方法.方法 先通过PCR对包含SNP位点的特异基因序列进行扩增;然后通过带有A标记的ASE引物针对SNP位点的不同基因类型进行特异性延伸;延伸后的产物可以与B标记的探针进行杂交结合,形成同时携带A和B标记的杂交产物;而该杂交产物可以通过核酸试纸条进行目视化检测,从而完成对SNP基因型的检测.结果 通过对10倍浓度梯度稀释的人类基因组DNA进行检测,PCR-ASE的检测敏感性为88 ng/反应;通过在同一条核酸试纸条上针对同一SNP位点2种不同基因型的检测,达到了多重检测的目的;PCR-ASE对19名志愿者的5个不同SNP位点的检测结果与基因测序法完全一致.结论 PCR-ASE 是一种简单、准确的SNP基因型检测方法.%Objective To establish polymerase chain reaction(PCR),allele-specific extension(ASE)and nucleic acid detection strip-based detection for single nucleotide polymorphisms(SNP).Methods The specific gene sequence containing SNP sites was amplified by PCR.For each SNP site,ASE primers labeled with A were extended.The ASE reaction products can bind to probe labeled with B,and hybridization products containing A and B simultaneously were formed.The products can be detected by disposable amplicon cross-contamination proof device containing a nucleic acid detection strip,and the detection of SNP genotypes can be accomplished.Results Ten-fold serial dilutions of quantified human genomic DNA were used to determine the sensitivity of PCR-ASE(88 ng/reaction).The ability of duplex PCR-ASE with the products can be detected by a single nucleic acid detection strip.A total of 19 samples representing 5 common SNP were detected by PCR-ASE,and the results had the consistency of 100％with DNA sequencing.Conclusions PCR-ASE is simple and accurate detection for SNP.