单管双向等位基因专一性扩增的单核苷酸多态分型的新方法

目的：建立一种基于等位基因专一性PCR原理的单核苷酸多态（single nucleotidepolymorphism,SNP)分型新方法：单管双向等位基因专一性扩增（single-tube bi-directional allele specificamplification,SB－ASA），并考察专一性引物的3’端第3位碱基不配对对特异延伸的影响。方法：一个PCR反应体系包含两个3’末端分别与SNP两个等位基因特异结合的引物，它们延伸方向相反，产生长度不同的等位基因专一扩增产物，同时在两个等位基因特异性引物的3’端第3位碱基引入不配对以增加特异性。PCR产物经琼脂糖凝胶电泳后分析确定样本的基因型。观察在不同的温度条件下，近3’末端引入与不引入碱基不配对时两种引物特异延伸的情况，比较两种引物能特异延伸的退火温度（annealingtemperature,Ta)范围。结果：对于4个不同类型的SNP位点，SB－ASA都成功地分型了36个样本，与直接测序的结果完全一致。两条专一性引物3’物第3位碱基引入不配对后，能特异延伸的退火温度Ta范围分别从64℃～69℃、60℃～62℃扩大到46℃～66℃、56℃～61℃。结论：SB－ASA是一种简单快速而有效的SNP分型新方法；在等位基因专一性PCR体系中，专一性引物3’端第3位碱基引入不配对能增加引物对两个等位基因的区分能力。

A simple and rapid new method for SNP typing by single-tube bi-directional allele specific amplification

OBJECTIVE: To establish a new method for single nucleotide polymorphism(SNP) typing based on allele specific PCR: single-tube bi-directional amplification (SB-ASA), and study the influence on specific extension by introducing a mismatch at the third 3'terminal base of allele specific primers. METHODS: Two allele specific primers, with a mismatch introduced at the third 3'terminal base, were both included in PCR system; they extended in opposite directions and amplified two allele specific fragments different in size. The genotype was determined by observing the length of amplified fragments after agarose electrophoresis. The proper ranges of annealing temperature (Ta) under which primers can specifically extend were achieved by observing the amplification status at different temperatures. RESULTS: SB-ASA was successfully used to type 36 samples for four different kinds of SNPs. Typing results were completely consistent with those by directional sequencing. Proper Ta ranges of two primers were expanded respectively from 64-69 degrees centigrade to 46-66 degrees centigrade and from 60-62 degrees centigrade to 56-61 degrees centigrade by introducing a mismatch at the third 3'terminal base. CONCLUSION: SB-ASA is a simple, rapid and efficient new method for SNP typing. During allele specific PCR reaction, specific primers with a mismatch at the third 3'terminal base have more power to identify two alleles.