基于等位基因特异性PCR原理建立的SNP分型新方法

目的建立一种新方法,对多个单核苷酸多态性(singlenucleotidepolymorphism,SNP)位点进行分型。方法基于等位基因特异性PCR原理,采用荧光标记复合扩增和毛细管电泳技术,根据PCR片段长度差异进行分型。选择SNP位点共11个,每个SNP位点设计两条长度不同、3’末端分别与SNP两个等位基因碱基配对的上游引物,同时为了增加特异性,在两条等位基因上游引物的3’末端第3或第4位碱基人为引入错配。在距离上游引物100～300bp范围内的合适位置,设计下游共用引物,并进行荧光标记。所有位点经过复合扩增后,PCR产物经ABIPrismTM310型遗传分析仪电泳分离,确定每个SNP的基因型。结果每个SNP位点纯合子为单一产物峰,杂合子则为长度不同的两个产物峰。不同的SNP位点扩增产物长度不同,根据产物长度和产物峰的数量进行SNP分型,一次完成11个SNP位点分型,其结果与直接测序完全一致。结论荧光标记复合扩增片段长度差异等位基因特异性PCR法是一种简单快速而有效的SNP分型新方法。

A new method for SNP typing based on allele specific PCR

OBJECTIVE: To establish a new method of SNP typing. METHODS: Based on the principle of allele specific PCR and capillary electrophoresis technique, 11 diallelic SNP loci were selected and two forward primers with different length were designed for each SNP, with their 3' ends matched to the two alleles, respectively. An artificially mismatched base was also introduced into the third or fourth base in the 3' end area of the two forward primers in order to enhance the specificity of amplification. A common reverse primer was designed 100-300 bp away from the forward primers, and labeled with fluorescence. The PCR products were separated and analyzed by ABI Prism 310 Genetic Analyzer after all of the 11 SNPs were multiply amplified. RESULTS: A single product peak was observed while the SNP was homozygous, and two product peaks with different height were observed while the SNP was heterozygous. The length of PCR products was different with the different SNPs. According to the length of the products and the number of the product peaks, the genotypes of the 11 SNPs can simultaneously be analyzed, and the results were in accordance with the direct sequencing. CONCLUSION: Fragment length discrepant allele specific fluorescence labeled multi-PCR (FLDASFLM-PCR) is a simple, rapid and efficient new method for SNP typing.