Since July 2004, Mitotyping Technologies has been amplifying and sequencing a approximately 150 base pair fragment of mitochondrial DNA (mtDNA) that codes for 12S ribosomal RNA, to identify the species origin of nonhuman casework samples. The approximately 100 base pair sequence product is searched at http://www.ncbi.nlm.nih.gov/BLAST and the species match is reported. The use of this assay has halved the number of samples for which no mtDNA results are obtained and is especially useful on hairs and degraded samples. The availability of species determination may aid forensic investigators in opening or closing off lines of inquiry where a highly probative but challenging sample has been collected.

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The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5'-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.

BackgroundMitochondrial DNA analysis is typically applied to degraded skeletal remains and telogen or rootless hairs. Data on the application of the method to very small hairs less than 0.5 cm from an age-matched and -challenged sample set are lacking.MethodsOne hundred fourteen hairs sized less than 1 cm from a 1993 case were analyzed for mitochondrial DNA according to laboratory standard operating procedures. For some hairs, a screening approach was applied, which permitted some samples, such as victim hairs on victim clothing, to be eliminated from the process quickly. Degraded samples were amplified with “mini-primers,” and 12S species testing was applied when non-human hairs were encountered.ResultsPartial to full control region human mitochondrial DNA profiles or species identifications (non-human species) were obtained from 93% of hairs under 1 cm, 92% of hairs under 5 mm, and 90% of hairs under 3.5 mm. Nineteen of 21 hairs 2 mm or less gave full or partial profiles. Among 128 hairs of all sizes tested in the case, 9 gave no results, 3 were canine in origin, and 73 did not exclude six known individuals tested in the case. Twenty-two hairs had nine additional profiles that were observed two or more times each. Twenty-one hairs showed singleton types not matching each other or any individual.ConclusionsCrime scene hairs that are both aged and small are often judged to be unsuitable for either hair microscopy or DNA analysis. This study of age-matched challenged small hairs indicates that even the smallest probative crime scene hairs are suitable for mitochondrial DNA analysis and can provide useful data.