Restriction Enzymes ApaI Analysis to Find A3243G Mutation in Indonesia Diabetes Mellitus Type II Patients

Abstract

The Use of PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) to find out the potential of mitochondrial DNA mutation at A3243G in type II Diabetic Patient has been done. Peripheral blood from 100 Indonesian type 2 diabetic subjects was selected randomly for this experiment. Peripheral blood lymphocyte was isolated, lysed, and it was in vitro amplified by PCR using a pair D1/D2 primers. PCR products were 294 base pair (bp) fragments which were then purified by ethanol precipitation method and characterized by restriction enzyme ApaI. Heteroplasmic A3243G mutation which was identified in 2 Subject (0,02%) was shown by 3 electrophoretic bands, 2 restriction products of APAI, i.e a 182 bp fragment and a 112 bp fragment; also a full fragment 294 bp, this means show that PCR-RFLP technique was approved for identifying heteroplasmic A3243g mutation in a tRNA gene mtDNA type 2 DM subject. 