建立以一对特异性引物选择性延伸测定SNP的方法及微流芯片仪器系统。该引物的3’端控制着引物的延伸反应，以微流芯片上测得的不同长度延伸产物确定SNP的类型。与改造的多重PCR技术相结合能同时测定多个SNP。并用本法实测糖尿病相关基因中的SNP。本研究可形成拥有自主知识产权的SNP微流芯片测定系统。可加快中国人群SNP与疾病及药物易感性关系的研究步伐。

In the post genome era, it is one of the main targets to clarify the relation between single nucleotide polymorphisms (SNPs) in whole genome sequence and susceptibility to disease or drugs. There is a demand for developing an SNP typing method with easy operation, no requirement of special instruments, and low running-cost. In this project, a single-tube genotyping method as well as a multiplexed SNP typing method were established using microchip electrophoresis as a detection platform, and following results were achieved. (1) A method for preparing micro chips and a detection system for microchip electrophoresis were set up especially for SNP typing. (2) An accurate and rapid SNP typing approach was developed by using extension reactions from two allele-specific primers in a single tube; and it was successfully employed for allele-frequency determination. (3) A method for typing SNP by extension reaction from anchored allele-specific primers and a universal primer was proposed. (4) A buffer enabling direct SNP genotyping using whole blood without the genome DNA extraction was prepared; and also filter paper-dried blood was successfully used as a starting material of SNP detection when the buffer was added in the detection mixture. This method extremely avoids the cross-contamination from sample preparation, sig

单碱基多态性（SNP）与疾病易感性和药物敏感性的关系研究是后基因组时代的重要内容之一，为了建立一种操作简便、无需特殊仪器和运行成本低的高通量SNP测定法，本课题采用微流体芯片为检测平台，建立了单管SNP测定法和多重SNP测定法。取得了如下结果：(1)制作了可供SNP测定的微流体电泳芯片制作和检测系统；（2）建立了一种在单管中进行双等位基因特异性引物延伸反应快速准确测定SNP的方法，并成功用于定量测定等位基因频率；（3）提出了锚定SNP特异性引物和一个通用引物延伸扩增法测定单碱基多态性的方法；（4）建立了不经提取血液中基因组DNA而直接进行SNP检测的方法，并成功采用滤纸血为起始材料进行SNP检测，极大避免了交叉污染，并使测定成本大大减低，也方便了样品采集；（5）提出采用酶切、DNA适配器技术与微流控芯片相结合同时测定多个SNP的方法（ALM-ASP），该法具有特异性高、样品用量少和操作简单等优点，在常规多重SNP测定中无需进行复杂条件优化就可以得到准确结果。实验表明55个SNP位点仅需八次测定就能完成，已成功用于测定857个样本，最多能达到17重SNP的同时测定。