关键词: 单核苷酸多态性, 高分辨率熔解曲线, 等位基因特异性引物延伸, 碱基对中性突变

Abstract: Objective To establish a modified high resolution melting assay based on allele-specific-extension(ASE-HRM)to determine single nucleotide polymorphisms(SNP).Methods The established ASE-HRM was designed as asymmetric polymerase chain reaction and melting curve analysis.An allele-specific-extension(ASE) primer which ends at the SNP site and matches one of the allele was added to the reaction system.Initially,asymmetric PCR was performed with undergoing additional thermal steps in order to get allele-specific-extension.A total of 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing.Results Heterozygotes could be discriminated from homozygotes according to their lower and broader melting transitions.Two different types of homozygote could be separated according to the presence or absence of an extension peak.Extension primers 11～13 bases long worked efficiently in an allele specific way.Modification of the limiting amplification primer with locked nucleic acid increased the Tm difference between extension and amplification peaks and facilitated subsequent genotyping.The 194 human genomic DNA samples were genotyped with the developed assay and by direct sequencing,with the different methods providing identical genotyping results.Conclusion ASE-HRM is a simple,inexpensive,closed-tube genotyping method that can be used to examine all types of SNP.

Key words: single nucleotide polymorphism, high resolution melting curve analysis, allele-specific-extension, base-pair neutral variant