高保真酶特异性检测大鼠SNP基因型新方法

摘要

Objective To establish a new SNP genotyping method using Pfu DNA polymerase extend phosphorothioate-modified primers in a single tube bi-directional allele specific amplification (SB-ASA). Methods For rat dbSNP loci rs8149053, we designed two outer and two inner allele-specific primers that were 3'-terminal phosphorothioate modified. Pfu DNA polymerase was used to specifically amplify the genome template and the SNP genotype was determined by different target segment DNA eletrophoresis. Results Different length segments represented different genotypes that were validated by sequencing. Conclusions Pfu polymerase genotyping assay is a rapid and specific method for detecting the genotype of the known SNP in a single PCR reaction.%目的 应用高保真酶(Pfu)和3'末端修饰引物在单管双向等位基因特异性扩增(SB-ASA)中区分SNP基因型,建立高保真酶特异性检测SNP基因型的新方法.方法 选取近交系大鼠SNP位点,以RS8149053为例,设计两个外部引物和两个等位基因特异性引物,四引物3'末端进行硫代磷酸化修饰,应用高保真聚合酶(Pfu)进行特异性扩增,扩增结果测序验证其可靠性.结果 在RS8149053 SNP位点(C/T)上,等位基因型CC扩增出179 bp目的片段,基因型TT扩增出597 bp目的片段,基因型不同则扩增出分子量不同的片段,目的条带测序结果与Rat Genome Database数据库基因型结果一致,高保真酶扩增结果稳定且特异性强.结论 高保真酶等位基因特异性扩增技术能有效降低假阳性率,是一种快速、特异的SNP基因分型新方法.