多重等位基因特异性扩增--微流控芯片电泳法同时测定多个单核苷酸多态性位点

Microchip electrophoresis coupled with multiplex allele-specific amplification for typing multiple single nucleotide polymorphisms(SNPs) simultaneously

文章以微流控芯片电泳为检测平台,建立了一种基于DNA适配器连接介导的多重等位基因特异性扩增同时测定多个单核苷酸多态性(SNP)位点的方法。以白细胞介素1β(IL1B)基因中的7个SNP位点(794C>T、1274C>T、2143T>C、2766T>del、3298G>A、5200G>A和5277C>T)为检测对象,通过PCR预扩增得一段含该7个待测SNP位点的长片段;用限制性内切酶MboⅠ将其消化成短片段,再与DNA适配器(adapter)相连;以连接产物为模板,在两管中分别用7条等位基因特异性引物和一条公用引物进行7重等位基因特异性扩增;最后用微流控芯片电泳法分离等位基因特异性扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。采用本法成功测定了48名健康中国人的IL1B基因上的7个SNP位点,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)和测序法测定结果完全一致。本法结果准确,可用于同时测定多个SNP位点;以微流控芯片电泳作为检测平台,分析速度快,样品需要量少;借助于自制筛分凝胶和重复使用芯片,使得SNP分析成本大大降低。

A new method of DNA adapter ligation-mediated allele-specific amplification （ALM-ASA） was developed for typing multiple single nucleotide polymorphisms （SNPs） on the platform of microchip electrophoresis. Using seven SNPs of 794C〉T, 1274C〉T, 2143T〉C, 2766T〉del, 3298G〉A, 5200G〉A, and 5277C〉T in the interleukin 1B （IL1B） gene as a target object, a long DNA fragment containing the seven SNPs of interest was pre-amplified to enhance the specificity. The pre-amplified DNA fragment was digested by a restriction endonuclease to form sticky ends; and then the adapter was ligated to either end of the digested fragment. Using the adapter-ligated fragments as templates, a 7-plex allele-specific amplification was performed by 7 allele-specific primers and a universal primer in one tube. The allele-specific products amplified were separated by chip electrophoresis and the types of SNPs were easily discriminated by the product sizes. The seven SNPs in IL1B gene in 48 healthy Chinese were successfully typed by microchip electrophoresis and the results coincided with those by PCR-restriction fragment length polymorphism and sequencing method. The method established was accurate and can be used to type multiple SNPs simultaneously. In combination with microchip electrophoresis for readout, ALM-ASA assay can be used for fast SNP detection with a small amount of sample. Using self-prepared gel matrix and reused chips for analysis, the SNP can be typed at an ultra low cost.