摘要
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
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°C-69°C to 46°C-66°C and from 60°C-62°C to 56°C-61°C 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.
出处
《中华医学遗传学杂志》
EI
CAS
CSCD
2001年第4期306-309,共4页
Chinese Journal of Medical Genetics
基金
国家自然科学基金重大项目 (398962 0 0 )
国家重点基础研究发展规划项目 (G1 9980 51 0 0 2 )&&
