New application for haplotype-specific extraction: Separation of mitochondrial DNA mixtures

A particularly challenging aspect of forensic casework is the accurate interpretation of samples that contain DNA from more than one contributor, since DNA analysis of specific identification markers often results in ambiguous mixed profiles. Physically separating the various haploid components of a diploid or polyploid DNA sample or of a specimen containing DNA from multiple individuals can resolve this problem. We adapted and combined previously developed methods of haplotype-specific extraction (HSE) to separate and analyze mitochondrial DNA (mtDNA) in samples containing DNA from two individuals. After optimizing conditions and probes, we performed HSE to separate mitochondrial DNA mixtures, which manifest as mixed nucleotide calls at single base positions. We were able to subsequently identify the DNA of the two individuals by sequencing. Based on our findings from this proof-of-concept experiment, this novel assay will be useful for distinguishing among the mtDNA of individuals in mixed DNA samples.