Validation of mitochondrial DNA minisequencing for forensic casework

The validation of multiplex solid-phase fluorescent minisequencing of mitochondrial DNA (mtDNA) for use in forensic casework is presented. Validation included testing of the reliability and species specificity of the technique, analysis of mixed body fluid samples, analysis of samples and substrate controls from previous cases and somatic stability of mtDNA. Animal, bacterial and fungal species extracts were examined and the test did not show cross-reactivity with other species. Hair, blood, saliva, faeces and semen or vaginal samples were tested from five male and five female individuals. For all the samples tested, heteroplasmy was observed only at position 302/309.1. Body fluid mixtures (blood:saliva, semen:saliva, faeces:semen, vaginal:semen) and DNA:DNA mixtures were examined. In total, 189 mixtures were analysed of which one resulted in a hybrid profile consisting of peaks from each of the two donors. The semen fraction of the semen:saliva and vaginal:semen mixtures appeared to be concentrated in the supernatant fraction of the extract thus highlighting the need to extract both the pellet and supernatant fractions of a stain. Control samples, crime stains and their substrate controls from previous cases were examined. Of the 12 loci typed by minisequencing, 11 could be verified by comparison to results from the sequencing method currently in use for casework and no discrepancies were observed between the two. MtDNA minisequencing was found to be a reliable and reproducible technique and its rapid and discriminating nature make it particularly suitable as a screening technique.