U.S. Department of Defense


Date of this Version



Final Report NIJ Grant # 2000-IJ-CX-K010. This document is a research report submitted to the U.S. Department of Justice.


This report details the work performed over a period of five years in an NIJ-funded project intended primarily to develop a practical means for accessing the large reserve of genetic variation in the ~15,000 bp mtDNA coding region, as a means for augmenting the forensic discrimination provided by sequencing the hypervariable (HV) regions of the mtDNA control region. Our approach was to focus on the relatively small number of particularly common types present in US Caucasian, African American, and Hispanic populations, where the problem of limited discrimination is concentrated in practice. We sequenced 506 entire mtDNA genomes corresponding to 56 common HV types present at 0.5% or more in the respective populations. Full genome sequencing resolved the 56 HV types into 423 haplotypes, and permitted the identification of 123 SNP sites suitable for practical assay development. Selection criteria for target SNPs included that they be present in multiple individuals, not be redundant with variation in the HV regions, and that they not occur at positions that affect either amino acid sequence or changes in structural RNA genes. Selected discriminatory SNPs were organized into panels that are specific for particular common HV types, or their near relatives. The intent was for a forensic scientist, encountering a common HV type, to be able to turn to one or two multiplex assays that represent the best chance for detecting additional discriminatory variation for the HV type in question. This was demonstrated to be a particularly effective strategy based on the observed distribution of variation in the mtDNA coding region. For Caucasian common HV types, eight multiplex allele-specific primer extension (ASPE) assays were designed, optimized, and tested. These proved to be highly suitable with regard to characteristics important for forensic mtDNA testing. Developmental validation for sensitivity, mixture detection, and degraded samples was completed for seven of the eight panels (one still in development). One multiplex has been applied in numerous case investigations, establishing a clear practical utility. Population databases were established for the completed multiplexes. Additional work on this project included generation of new control region databases, including four regional Hispanic population samples that were significantly differentiated at the haplogroup and haplotype level. Also developed, in a collaborative project, was a multiplex ASPE SNP assay for haplogroup assignment among mtDNAs of W. European origin.