一种基于磁性颗粒和通用连接子扩增技术的单核苷酸多态性分型方法

Magnetic Particle-based Single Nucleotide Polymorphism Detection Method Using Linker-Polymerase Chain Reaction

提出了一种应用磁性颗粒和通用连接子扩增技术(Linker-PCR)的多位点单核苷酸多态性(SNP)分型方法.该方法首先通过酶切将样本基因组DNA打断,然后将通用连接子通过T4DNA连接酶与各个酶切片段连接,利用生物素标记的通用引物将样本进行全基因组扩增.扩增后,将生物素标记的Linker-PCR扩增产物固定到亲合素修饰的磁性颗粒表面,通过与双色荧光标记的等位基因特异性探针杂交,对待测位点进行分型.利用该方法,我们对10个样本MTHFR基因上的2个SNP位点进行了分型,分型结果准确、正错配信号比大于3.由于利用Linker-PCR技术来实现对靶序列的全基因组扩增,该方法非常适用于大量样本的多基因多位点的SNP分型研究.

An approach was presented to type multiplex single nucleotide polymorphism （SNP） by means of magnetic particles and amplification of whole genome products using universal linker-PCR. The linker-PCR products were captured by magnetic particles modified with streptavidin to fabricate SNP li- brary. Each SNP locus in the library was interrogated by hybridization with a pair of dual-color fluorescence （Cy3, Cy5） probe, and the genotypes could be simultaneously identified by scanning the microarray printed with the denatured fluorescent probes on an unmodified glass slide. This method was applied to analysis the methylenetetrahydrofolate reductase （MTHFR） gene C677T and A1298C polymorphisms for ten different samples. The fluorescent ratios （match/mismatch signal） of homozygous samples were over 3. This simple and reliable genotyping method is suitable for high-throughput genotyping for multiplex loci, and will find a wide potential application to research and medical diagnosis.