Journal article
Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality
Forensic Science, Medicine and Pathology, Vol.14(1), pp.70-75
2018
Abstract
Mitochondrial DNA (mtDNA) can provide a means for forensic identity testing when genotyping of nuclear DNA (nuDNA) targets is not possible due to degradation or lack of template. For degraded samples, an indication of the quantity and quality of mtDNA is essential to allow selection of appropriately sized targets for hypervariable region (HVR) analysis, which may conserve sample and resources. Three human-specific mtDNA targets of increasing length (86, 190 and 452 base pairs) were amplified by singleplex quantitative real-time PCR (qPCR), capable of providing an index of mtDNA degradation from fragment length information. Quantification was achieved by preparation of a standard curve for each target, using a purified mtDNA standard containing all three targets of interest, which produced a linear, accurate and precise result from 1×108 to 10 copies. These novel assays demonstrated excellent sensitivity, specificity and reproducibility in line with the minimum information for qPCR experiments (MIQE) guidelines. Further, a separate inhibition control reaction was included to guide sample clean-up and ensure the validity of degradation assays. This protocol assists the selection and analysis of appropriately sized targets to maximize the chance of obtaining an informative result in downstream assays like sequencing.
Details
- Title
- Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality
- Authors/Creators
- C. Goodwin (Author/Creator) - University of CanberraD. Higgins (Author/Creator) - The University of AdelaideS.S. Tobe (Author/Creator) - ArcadiaJ. Austin (Author/Creator) - The University of AdelaideA. Wotherspoon (Author/Creator) - Australian Nuclear Science and Technology OrganisationM.E. Gahan (Author/Creator) - University of CanberraD. McNevin (Author/Creator) - University of Canberra
- Publication Details
- Forensic Science, Medicine and Pathology, Vol.14(1), pp.70-75
- Publisher
- Springer US
- Identifiers
- 991005540499007891
- Copyright
- © 2018 Springer Science+Business Media, LLC, part of Springer Nature
- Murdoch Affiliation
- Murdoch University
- Language
- English
- Resource Type
- Journal article
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- Domestic collaboration
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- Citation topics
- 1 Clinical & Life Sciences
- 1.189 Genome Studies
- 1.189.310 Population Genetics
- Web Of Science research areas
- Medicine, Legal
- Pathology
- ESI research areas
- Clinical Medicine