Evaluation of novel mitochondrial DNA panels for forensic uses

Publication Type:
Thesis
Issue Date:
2018
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Mitochondrial DNA testing is expanding the use of DNA as a forensic tool for human identification. The testing of mitochondrial DNA is a common practice for biological samples, which are compromised, degraded and contain limited STR information. In these cases, the amplification and sequencing of mitochondrial DNA becomes a valuable tool in determining the source of DNA samples. A review of mitochondrial DNA testing in forensic science reveals a number of improvements which can be made to this process. The project therefore aimed to improve the process of mitochondrial DNA testing. In particularly, it focuses on the developing and testing of mitochondrial DNA markers involved in the quantification and amplification of samples. The quantification of mitochondrial DNA is important to optimising the amplification of DNA samples. Methods for quantification of mitochondrial DNA commonly involve estimating mitochondrial DNA quantities from nuclear DNA. This is usually unreliable as a high variability of mitochondrial DNA copy number exists between human cells. Hence, in this study, the development of a specific DNA assay for mitochondrial DNA provides a reliable determination of mitochondrial copy number. The testing of this novel assay has shown it is specific, sensitive and reproducible in DNA samples of artificially degraded qualities. As such, the incorporating of the DNA assay into workflows of mitochondrial DNA testing will improve the overall amplification of samples for downstream processes such as mitochondrial DNA sequencing. Furthermore, the transition of mitochondrial DNA sequencing from capillary electrophoresis to massively parallel sequencing platforms is increasing the feasibility of typing multiple DNA fragments in a single reaction. This has led to the development of small PCR markers, which are capable of amplifying the entire mitochondrial genome even in challenging forensic samples. While the sequences of these PCR markers and panels are available for use, its full performance in amplifying compromised samples remains unknown due to the limited and usually specialist use of mitochondrial DNA testing and massively parallel sequencing in forensic laboratories. Therefore, the technical work carried out in this study tests the performance of the Early Access AmpliSeq™ Mitochondrial Panel (Applied Biosystems, CA, USA) for amplifying complete mitochondrial genomes in samples of degrading qualities. The testing of this Panel in compromised samples with limited STR success informs the use of the Panel in the mitochondrial testing of DNA in forensic laboratories. In this study, the typing of amplified DNA fragments in parallel reveals the recovery of complete mitochondrial DNA sequences in all samples. These samples were concordant to reference sequences and the HV1 and HV2 sequences provided by the ‘gold standard’ of capillary electrophoresis platforms. Importantly, the analysis of mitochondrial DNA sequences shows a capability to resolve mitochondrial haplogroups and ancestries for familial matching. Overall, the results of this technical work confirms the Panel is able to amplify the complete mitochondrial genome of compromised samples for sequencing using massively parallel technologies. As such, this contributes to the use and validation of massively parallel sequencing technologies in forensic DNA testing. Overall, the development of a novel DNA assay for the quantification of mitochondrial DNA and the technical testing of the Early Access AmpliSeq™ Mitochondrial Panel (Applied Biosystems, CA, USA) has contributed to improvement of mitochondrial DNA testing in compromised samples. Past use of mitochondrial DNA testing has provided identifications in cases of mass disasters, missing persons and historical remains. As such, the improvements to this process will continue to assist in these identifications.
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