文章摘要
汪维鹏,李 融,王 森.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[J].南京医科大学学报,2009,29(4):576~581
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
Multiplex SNP typing using monotube allele-specific amplification
投稿时间:2008-11-12  
DOI:10.7655
中文关键词: 等位基因特异性扩增  单核苷酸多态性  单管  多重PCR  TYR
英文关键词: allele-specific amplification  SNP  monotube  multiplex PCR  TYR
基金项目:苏州大学青年教师自然科学基金资助项目(Q3132819);苏州大学大学生创新性实验计划项目(57315853和57315488)
作者单位
汪维鹏 苏州大学药学院,江苏 苏州 215123 
李 融  
王 森  
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中文摘要:
      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TYR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TYR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?
英文摘要:
      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 performed by using all allele-specific primers and a universal primer in a single tube by using the adapter-ligated DNA as templates. Finally,the allele-specific amplification products were separated by agarose gel electrophoresis,and the types of the SNPs were discriminated by the length of the amplified products. Results:Three SNPs in the TYR gene were successfully typed for 30 healthy Mainland Chinese and the results were in agreement with those obtained by RFLP. Conclusion:This method with high specificity is accurate and cost-effective,and can be used for typing multiple SNPs simultaneously.
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