GENOTYPING METHOD AND FREQUENCY OF SINGLE NUCLEOTIDE POLYMORPHISM RS12970134 NEAR MC4R GENOTYPES IN VIETNAM PRESCHOOL CHIDREN POPULATION

Abstract

Melanocortin-4 receptor is part of the central melanocortinergic system and plays critical roles in central regulation of food intake, energy homeostasis and body weight, so that this gene is related to obesity and insulin resistance including single nucleotide polymorphism rs12970134. Thus, this study aimed to find out a protocol for genotyping rs12970134 near Melanocortin-4 receptor by AS-PCR method, analyzing the genotype and allele ratios of this single nucleotide polymorphism in 200 3-5 years old children in Hanoi, Vietnam (50% boys). This protocol used the forward primers including 5’-tcttaccaaacaaagcatgtg-3’ to detect allele G, and 5’-tcttaccaaacaaagcatgta-3’ to detect allele A; and the reverse primer 5’-gtcattcccactaccacctg-3’. The optimized PCR protocol was that 94^oC for 3 min and 34 cycles of denaturation at 94^oC for 30 sec, primer annealing at 54^oC for 40 sec, primer extension at 72^oC for 30 sec, final extension at 72^oC for 8 min, stopped by chilling at 4^oC. The 208 bp PCR products were detected on Redsafe-stained 2.5% agarose gel. The results were verified by using the sequencing method. In the entire samples, the GG genotype was the largest (57.5%), and the AA genotype was the lowest (4%). The frequencies of the G and A alleles were 0.77 and 0.23, respectively.