Restriction fragment length analysis of mitochondrial DNA for screening purposes in human identification

Human mitochondrial DNA (mtDNA) becomes extremely important for identity testing when genomic DNA is in insufficient quantity or compromised due to decomposition, fin, environmental insults, or aging. The hypervariable (HV) regions of the mitochondrial genome are the most polymorphic and therefore the most informative when identity testing is necessary. A preliminary study was conducted in order to determine if restriction enzyme digestion of these HV regions could generate sufficient information for screening mitochondrial DNA samples prior to more sophisticated analysis. DNA was extracted from blood stains obtained from 80 non-related individuals using the Chelex(R) method. The 1.3 kilobase (kb) control region was amplified, using the polymerase chain reaction (PCR) method, and the 2 HV regions were subsequently amplified using nested PCR. These two regions were then tested with 22 different restriction enzymes and the resulting products were visualized on agarose gels. Results show that a battery of 5 restriction enzymes (HinfI, KpnI MboI TaqI, RsaI) demonstrated significant variation in HV region I. Almost half, 44%, of samples could be screened into specific groups by digestion in HV1. HV region 2 did not show significant polymorphism with any of the 22 restriction enzymes tested. The results and applications are discussed.