荧光标记LDR-PCR复合扩增方法对高度降解DNA检材的SNPs分型研究

Study on SNP Genotyping of Degraded DNA by Fluorescence-labeled Multiplex LDR-PCR Amplification

目的构建一套基于连接酶检测反应(LDR)的荧光标记PCR复合扩增体系,为解决高度降解DNA法医学分析提供新的策略。方法选择8个单核苷酸多态性(SNPs)位点(rs10802248、rs10516197、rs10488372、rs2278945、rs4757318、rs4887255、rs4889002和rs9304473),设计合成各SNPs位点的LDR探针和连接产物PCR引物,通过LDR将连接产物进行PCR扩增,并将扩增产物进行毛细管凝胶电泳,构建复合扩增SNPs分型体系。结果使用荧光标记LDR-PCR复合扩增方法,对不同个体的8个SNPs位点进行分型,分型结果与测序结果完全一致;对于甲醛固定石蜡包埋组织(FFPET)检材高度降解的DNA样品,荧光标记LDR-PCR复合扩增体系能够实现8个SNPs位点的准确分型。结论荧光标记LDR-PCR复合扩增方法能够对8个SNPs位点进行一次性分型,结果准确、可靠,是一种简单、高效并且较为实用的SNPs分型新方法,适用于高度降解检材的检测。

Objective In this study, a multiplex PCR amplification system was constructed based on fluorescent labeling PCR and IX）R, to pro- vide a new strategy for analyzing severely degraded DNA. Methods Eight SNP loci （rs10802248, rs10516197, m10488372, rs2278945, rs4757318, rs4887255, rs4889002, and rs9304473 ） were selected. Their LDR probes and PCR primers of linked products were designed and syn- thesized. Ligase detection reaction, PCR amplification, and capillary gel electmphoresis （CEG） were performed to establish the multiplex LDR- PCR amplification system. Results The genotypes of these 8 loci were obtained simultaneously by the fluorescence-labeled multiplex LDR-PCR amplification method. The loci prot＇des obtained by fluorescence-labeled multiplex LDR-PCR amplification were in accordance with those obtained by direct sequencing of the pelymorphic regions in samples from all individuals. By fluorescence-labeled multiplex LDR-PCR amplification, the 8 SNP loci were efficiently amplified from the severely degraded FFPET DNA. Conclusion Eight SNP loci results could be obtained simultaneous- ly by using the multiplex LDR-PCR amplification system, which is a simple, efficient, and practical SNP genotyping method with accurate and reli- able results for highly degraded samples.