高保真酶特异性检测大鼠SNP基因型新方法

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。

Establishment of a New SNP Genotyping Method with Pfu DNA Polymerase Assay in the Rat

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 ...