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

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