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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性扩增产物 ,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′端第 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 6 4℃～ 6 9℃、6 0℃～ 6 2℃扩大到 46℃～ 6 6℃、5 6℃～ 6 1℃。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
Objective To establish a new single nucleotide polymorphism (SNP) typing method based on the principle of allele-specific PCR: single-tube bi-directional allele specificamplification, SB-ASA) and investigate the influence of unpaired base 3 ’on the 3’ end of the specific primer on the specific extension. Methods A PCR reaction system consisted of two primers specifically binding to two alleles at the 3 ’end of the SNP, respectively. They extended in the opposite direction and produced allele-specific amplification products of different lengths. At the same time, two alleles The third base of the 3 ’end of the specific primer is introduced unpaired to increase the specificity. The PCR product was analyzed by agarose gel electrophoresis to determine the genotype of the sample. To observe the extension of the two primers at the near 3 ’end under unpaired and non-introduced bases under different temperature conditions, and compare the range of annealingtemperature (Ta) between the two primers. Results For the 4 different types of SNP loci, SB-ASA successfully sequenced 36 samples, which is consistent with the results of direct sequencing. The temperature range of annealing temperature Ta that can be extended specifically from the third base at the 3 ’end of the two specific primers is extended from 46 ℃ to 69 ℃ and from 60 ℃ to 62 ℃ to 46 ℃ to 6 ℃ 6 ℃, 5 6 ℃ ~ 6 1 ℃. Conclusion SB-ASA is a simple, rapid and effective method for SNP typing. In the allele-specific PCR system, the introduction of the third base at the 3’-end of a specific primer can increase the number of primers Allele