Analysis of polymorphisms and heteroplasmy in mitochondrial DNA for human identification

Mitochondrial DNA has proven to be a useful target for analysis of highly degraded and limited DNA samples often encountered in forensic cases. A sensitive duplex PCR and probe based assay targeting 18 polymorphic sites within the hypervariable regions I and II (HVI/II) of the mitochondrial genome was developed and validated. This assay was also tested on forensically relevant samples and populations and was shown to be a useful screening tool for forensic casework samples and human identification. The HVI/HVII assay was also shown to have a relatively high discrimination power for the African American (0.993) and Japanese populations and somewhat lower in the US Caucasian and US Hispanic (0.945) populations. Several common HVI/HVII mitotypes were observed which resulted in the lower discrimination power, specifically in the US Caucasian and Hispanic populations. To further increase discrimination power of the assay, additional polymorphic sites distributed throughout the mitochondrial genome were identified and probes and primers were added to this assay. This 83 probe HV+ assay was shown to be highly discriminating and was found to be more informative than sequencing the HVI/HVII regions for the US Caucasian sample population.;A mixture of mtDNA sequences or heteroplasmy resulting from an insertion/deletion or point mutation may also be encountered in a forensic case sample. Heteroplasmy was characterized in different tissues and across age groups by sequence analysis. Length heteroplasmy was frequently observed in the HVI/HVII poly-C stretches across all tissues. The number of cytosines observed in the HVII C-stretch varied between tissues and a repeat of 7 C's was found to be more stable than 8 C's. Point heteroplasmy was observed at some positions more frequently than others (heteroplasmic hotspots) and occurred near the origins of replication. Point heteroplasmy was also more frequently observed in muscle tissue compared to other tissues analyzed, with the lowest in blood. Point heteroplasmy was shown to increase with age while length heteroplasmy was independent of age. The assays developed and described here were shown to be useful for detection of sequence polymorphisms and heteroplasmy in the human mitochondrial genome.