Resolution of mixed human DNA samples using mitochondrial DNA sequence variants

Abstract

Forensic casework samples routinely contain DNA from multiple contributors [1], posing a challenge to investigators attempting to resolve the components of complex DNA mixtures. Analysis of human mitochondrial DNA (mtDNA) is commonly used in forensic investigations to match evidentiary samples to potential suspects [2,3]. Unique DNA sequences from the hypervariable sequence (HVS) region of the mitochondrial genome (nucleotides 16,024–16,576) from different samples can be compared with known samples to determine if there is a ‘‘match.’’ However, this comparison strategy becomes exponentially complicated with the presence of additional contributors within human DNA samples. Recently, a technique based on denaturing high-performance liquid chromatography was reported for the rapid screening of mtDNA for resolution of mtDNA mixtures and the determination of the number of contributors [4]. Using this approach, ‘‘identity versus nonidentity’’ was accurately determined in less than 7min per sample for 106 pairwise comparisons. Although this approach demonstrates the ability to detect multiple sequences in a mixed sample, and subsequent pairwise comparisons can be used to identify potential contributors, the sequence data themselves are not obtained and it relies on specialized instrumentation that is not routinely available in most forensic laboratories. In addition to forensic applications, sequence analyses of the human mtDNA hypervariable control region have been performed by many investigators as a means of studying the demographic expansion and migration