单管等位基因特异性扩增法同时测定多重单核苷酸多态性

目的:建立了一种单管等位基因特异性扩增法同时测定多重单核苷酸多态性(SNP).方法:以TYP基因外显子1上的3个SNP位点(71G>A、425A>T和758G>A)为例,首先采用PCR预扩增得一段含所有待测SNP位点的长片段;然后用限制性内切酶将其消化成短片段,在连接酶的作用下与设计的DNA适配器相连;以此连接产物为模板,在单个PCR管中加入一种适配器特异性通用引物和所有等位基因特异性引物进行PCR扩增;最后用琼脂糖凝胶电泳法分离检测PCR扩增产物,并根据扩增片段的大小判断SNP的类型.结果:采用该法成功测定了30名健康中国人的TYP基因中的3个SNP位点,与限制性片段长度多态性法(RFLP)测定结果完全一致.结论:该方法特异性高、结果准确、检测成本低,可用于同时测定多个SNP位点.

Multiplex SNP typing using monotube allele-specific amplification

Objective:To establish mono-tube allele-specific amplification for typing multiplex SNPs simultaneously.Methods:Taking three SNPs of 71G > A,425A > T,and 758G > A in the TYR gene as example,a PCR pre-amplification was firstly carried out for producing a long target containing all SNPs of interest.Secondly,the preamplified DNA fragments were digested by a restriction endonuclease to form sticky ends,which were then ligated to a designed DNA adapter by ligase.Thirdly,an allele-specific amplification was perfo...