双向等位基因特异性PCR快速区分纯合子和杂合子SNP分型的新方法

Bidirectional PCR Amplification of Specific Alleles:A Novel Method of SNP Typing for Rapidly and Simultaneously Differentiating Homozygotes and Heterozygotes

目的建立一种基于单碱基改变基因分型的快速检测等位基因特异性双向PCR原理的改良SNP分型新方法:双向等位基因特异性PCR(Bi-PASA)在一次PCR反应中区分纯合子和杂合子的方法。方法以SNP位点rs11293201和rs57148397为例,设计两个外部引物(F与R)和两个内部等位基因特异性引物(P与Q)。同时与测序方法对比检测突变。结果两个内部引物(P和Q)包含一个相对短的完全匹配区域和一个富含GC的10核苷酸5′尾。当基因组DNA被先期扩增出的模板DNA取代时内引物完成从低效扩增到高效扩增的转变,其结果与直接测序完全一致。利用Bi-PASA参数的优化在人类SIP1基因常见突变的外显子中详细研究先天性巨结肠病,3种额外的Bi-PASA分析被快速优化。结论Bi-PASA是一种简单快速而有效的SNP分型新方法,是在一个PCR反应中检测已知突变的方法。

Objective Bidirectional PCR amplification of specific alleles （Bi-PASA） is a rapid method for genotyping single-base changes, but one reaction is required for each allele. This study was aimed to develop a new Bi-PASA method for single nucleotide polymorphis （SNP） typing to distinguish homozygotes and heterozygotes in one PCR reaction by utilizing novel primer design with appropriate cycling conditions. Methods For SNP loci rs11293201 and rs57148397, two outer （F and R） and two inner allele-specific （P and Q） primers were designed. Simultaneously, with comparison to sequencing, mutations were detected. Results The two inner primers （F and Q） contained a relatively short complementary region and a 10-nucleotide G ＋ C-rich 5′ tail. The inner primers switched from low-efficiency to high-efficiency amplification when genomic DNA was replaced by previously amplified template DNA. Typing results were completely consistent with those by direct sequencing. In addition, the 5 ＇ tails prevent ＂megapriming＂ The parameters for optimizing Bi-PASA were investigated in detail for common mutations in the human SIP1 genes on Hirschsprung Disease （HSCR） . Three additional Bi-PASA assays were rapidly optimized. Conclusion Bi-PASA is a simple, rapid and efficient new method for SNP typing, particularly for detecting known mutations in a single PCR reaction.