Design and Optimization of PCR-RFLP Assay for Detection of G9053A and T15663C Mutation in Mitochondrial DNA

Abstract

Mutation of mitochondrial DNA (mtDNA) such as G9053A and T1566C is involved in type 2 diabetes mellitus. The G9053A mutation in the ATP6 gen and the T15663C in the CYB gene has been reported to cause structure changes and interfering the respiration process in mitochondria. However, diagnosis method of type 2 diabetes mellitus by measuring blood sugar level is unable to detect point mutations in mtDNA. PCR has been extensively used for amplification of DNA sequences. Genetic analysis by restriction fragment length polymorphism (RFLP) is one of the most common methods used to examine nucleic acids for the presence of point mutation. In this study, PCR-RFLPs were designed and optimized in order to diagnose type 2 diabetes mellitus by detecting G9053A and T15663C mutation. The PCR primers forward and reverse were designed to amplify target gene by the PCR reaction. The DNA template was isolated from urinary epithelial cells of type 2 DM patients and then amplified through the PCR process. The PCR products were digested using the restriction enzymes HhaI and BccI and then characterized using agarose gel electrophoresis. Based on the results of electrophoresis, optimized condition of PCR-RFLP method was achieved as proved by the presence of 142 and 87 bp long bands for G9053A mutation and 129 and 95 bp long bands for T1566C mutation.