Rapid genotyping of APOA5 − 1131T>C polymorphism using high resolution melting analysis with unlabeled probes

The APOA5 − 1131 T/C polymorphism (rs662799) exhibits a very strong association with elevated TG levels in different racial groups. High resolution melting (HRM) analysis with the use of unlabeled probes has shown to be a convenient and reliable tool to genotyping, but not yet been used for detecting rs662799 polymorphism. We applied the unlabeled probe HRM analysis and direct DNA sequencing to assay the − 1131T>C SNP in 130 cases DNA samples blindly. This HRM analysis can be completed in < 3 min for each sample. The two melting peaks were displayed at 66.1 ± 0.4 °C for CC homozygote and 68.7 ± 0.2 °C for TT homozygote; TC heterozygote showed the both melting peaks. The genotyping results by HRM method were completely concordant with direct DNA sequencing. The distribution of CC, TC, and TT genotypes for the − 1131T>C SNP was 9.2, 49.2, and 41.5%, respectively. This assay was sensitive enough to detect C allele down to 20% and 10% for T allele. The limit of detection for C and T allele was 6.2 and 2.5 ng/μL DNA, respectively. The developed unlabeled probe HRM method provides an alternative mean to detect ApoA5 − 1131T>C SNP rapidly and accurately.

► We developed an unlabeled probe HRM analysis to assay the APOA5 − 1131 T/C polymorphism (rs662799).
► This assay was sensitive enough to detect C allele down to 20% and 10% for T allele.
► The limit of detection for C and T allele was 6.2 and 2.5 ng/μL DNA, respectively.