Primary study of SNP rs2046210 in Vietnamese breast cancer population by high-resolution melting analysis (HRMA)

Abstract

The single nucleotide polymorphism (SNP) rs2046210 (G/A allele) has been identified to be highly associated with breast cancer in Asian populations, especially Chinese (p = 4.1 × 10−5) and Japanese (p = 0.05). The association between SNP rs2046210 and breast cancer development tends to be examined using High resolution melting (HRM) analysis to genotype this SNP on Vietnamese DNA samples. In this study, optimizations of HRM key components such as primers, DNA, MgCl2 and DMSO concentrations were carefully performed for distinctive melting patterns between different genotypes. Positive controls were obtained through HRM analysis on several random samples and confirmed by direct sequencing. Our result indicated that 0.2 μM of primer was the best concentration to satisfy certain requirements, including obtaining substantial PCR products and appropriate CT value. For DNA concentrations, 10–50 ng were evaluated for their influence on PCR amplification, melting profile, and allelic discrimination of HRMA and the amount of 10 ng DNA was chosen as standard input for HRMA. Optimization results also revealed that the higher DMSO concentration was, the lower the amplicon’s Tm got. On the contrary, the effect of MgCl2 concentration on PCR reaction was to increase melting temperature. Finally, our developed optimal HRM protocol was successful for genotyping SNP rs2046210 for Vietnamese patients.