Study on SNP Genotyping of Degraded DNA by Fluorescence-labeled Multiplex LDR-PCR Amplification / 中国医科大学学报

ABSTRACT

Objective In this study,a multiplex PCR amplification system was constructed based on fluorescent labeling PCR and LDR,to provide a new strategy for analyzing severely degraded DNA.Methods Eight SNP loci (rs10802248,rs10516197,rs10488372,rs2278945,rs4757318,rs4887255,rs4889002,and rs9304473) were selected.Their LDR probes and PCR primers of linked products were designed and synthesized.Ligase detection reaction,PCR amplification,and capillary gel electrophoresis (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 profiles obtained by fluorescence-labeled multiplex LDR-PCR amplification were in accordance with those obtained by direct sequencing of the polymorphic 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 simultaneously by using the multiplex LDR-PCR amplification system,which is a simple,efficient,and practical SNP genotyping method with accurate and reliable results for highly degraded samples.