Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality

Goodwin, C; Higgins, D; Tobe, SS; Austin, J; Wotherspoon, A; Gahan, ME; McNevin, D

Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality

Goodwin, C Higgins, D Tobe, SS Austin, J Wotherspoon, A Gahan, ME McNevin, D

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Publication Type:: Journal Article
Citation:: Forensic Science, Medicine, and Pathology, 2018, 14 (1), pp. 70 - 75
Issue Date:: 2018-03-01

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Field	Value	Language
dc.contributor.author	Goodwin, C	en_US
dc.contributor.author	Higgins, D	en_US
dc.contributor.author	Tobe, SS	en_US
dc.contributor.author	Austin, J	en_US
dc.contributor.author	Wotherspoon, A	en_US
dc.contributor.author	Gahan, ME	en_US
dc.contributor.author	McNevin, D https://orcid.org/0000-0003-1665-3367	en_US
dc.date.available	2017-12-07	en_US
dc.date.issued	2018-03-01	en_US
dc.identifier.citation	Forensic Science, Medicine, and Pathology, 2018, 14 (1), pp. 70 - 75	en_US
dc.identifier.issn	1547-769X	en_US
dc.identifier.uri	http://hdl.handle.net/10453/131006
dc.description.abstract	© 2018, Springer Science+Business Media, LLC, part of Springer Nature. 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.	en_US
dc.relation.ispartof	Forensic Science, Medicine, and Pathology	en_US
dc.relation.isbasedon	10.1007/s12024-017-9944-8	en_US
dc.subject.classification	Legal & Forensic Medicine	en_US
dc.subject.mesh	Humans	en_US
dc.subject.mesh	DNA, Mitochondrial	en_US
dc.subject.mesh	RNA, Ribosomal	en_US
dc.subject.mesh	RNA, Ribosomal, 16S	en_US
dc.subject.mesh	DNA Primers	en_US
dc.subject.mesh	Electrophoresis, Agar Gel	en_US
dc.subject.mesh	DNA Degradation, Necrotic	en_US
dc.subject.mesh	Real-Time Polymerase Chain Reaction	en_US
dc.title	Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality	en_US
dc.type	Journal Article
utslib.citation.volume	1	en_US
utslib.citation.volume	14	en_US
utslib.for	0699 Other Biological Sciences	en_US
utslib.for	0604 Genetics	en_US
utslib.for	1103 Clinical Sciences	en_US
pubs.embargo.period	Not known	en_US
pubs.organisational-group	/University of Technology Sydney
pubs.organisational-group	/University of Technology Sydney/Faculty of Science
pubs.organisational-group	/University of Technology Sydney/Faculty of Science/School of Mathematical and Physical Sciences
pubs.organisational-group	/University of Technology Sydney/Strength - CFS - Centre for Forensic Science
utslib.copyright.status	closed_access
pubs.issue	1	en_US
pubs.publication-status	Published	en_US
pubs.volume	14	en_US

Abstract:

© 2018, Springer Science+Business Media, LLC, part of Springer Nature. 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.

Please use this identifier to cite or link to this item:

http://hdl.handle.net/10453/131006