The Development of Multiplex PCR-RFLP of FTO Genetics Variants

Abstract

Background: Major health concern throughout the whole world is the increasing rate of obesity, either in elder group or younger group of multiracial ethnics that are believed to be associated with genetic factor that plays important role in the pathogenesis of obesity. The very first gene to be associated with obesity is fat mass and obesity-associated (FTO) gene with the involvement from many single nucleotide polymorphisms (SNPs) in the first intron of FTO. Early detection of obesity-related SNPs is very crucial in the effort of reducing obesity rate which is expected to increase by 2025.This has led to the urge of developing a method to detect individual with predisposing factors towards obesity, with the hope that this method will eventually helpful in assisting healthcare authorities to rapidly detect individual who are at high risk of being obese. Multiplex PCR-RFLP is a method for rapid detection of obesity-related SNPs using gene sequence amplifying technique and optimization done using gradient PCR technique. Thus, this study was conducted to evaluate the potential of multiplex PCR-RFLP to amplify FTO genetic variants. Materials and methods: A total of 14 UniSZA students were recruited to obtain 2 ml of peripheral whole blood for DNA extraction. Single gradient PCR dan multiplex PCR were employed to determine the optimum annealing temperature for all assigned FTO SNPs as rs9939609, rs1421085 and rs17817288. The PCR products were run on 1% of agarose gel electrophoresis and visualized under UV Gel doc Image Analyzer. These PCR products were introduced to restriction enzymes for the cutting site on genetic variation namely Bfal, HpyCH4V, HpyCH4IV for rs9939609, rs1421085 and rs17817288, respectively. Next, the PCR fragments were identified on 2% agarose gel electrophoresis under UV Gel Doc Image Analyzer. Results: The optimum annealing temperature to amplify all FTO SNPs obtained through gradient PCR was 60°C. The amplification of FTO rs9939609, rs17817288 and rs1421085 regions, showed specific size product of 151 bp, 140 bp, and 131 bp respectively. In RFLP, the fragments after restriction enzyme digestion produced homozygous wild type, heterozygous and homozygous mutant type for genotype characterization based on allele cut site by specific restriction enzyme namely BfaI, HpyCG4IV and HpyCH4V, respectively. Conclusion: This study may become preliminary data on the potential of multiplex PCR-RFLP in provide rapid diagnosis to amplify multiple FTO genetic variants.