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

Corey Goodwin, Denice Higgins, Shanan S. Tobe, Jeremy Austin, Andrew Wotherspoon, Michelle E. Gahan, Dennis McNevin

    Research output: Contribution to journalArticle

    2 Citations (Scopus)

    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.

    Original languageEnglish
    Pages (from-to)70-75
    Number of pages6
    JournalForensic Science, Medicine, and Pathology
    Volume14
    Issue number1
    DOIs
    Publication statusPublished - 1 Mar 2018

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    Mitochondrial DNA
    Real-Time Polymerase Chain Reaction
    Base Pairing
    Guidelines
    Sensitivity and Specificity
    DNA

    Cite this

    Goodwin, Corey ; Higgins, Denice ; Tobe, Shanan S. ; Austin, Jeremy ; Wotherspoon, Andrew ; Gahan, Michelle E. ; McNevin, Dennis. / Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality. In: Forensic Science, Medicine, and Pathology. 2018 ; Vol. 14, No. 1. pp. 70-75.
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    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.",
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    Singleplex quantitative real-time PCR for the assessment of human mitochondrial DNA quantity and quality. / Goodwin, Corey; Higgins, Denice; Tobe, Shanan S.; Austin, Jeremy; Wotherspoon, Andrew; Gahan, Michelle E.; McNevin, Dennis.

    In: Forensic Science, Medicine, and Pathology, Vol. 14, No. 1, 01.03.2018, p. 70-75.

    Research output: Contribution to journalArticle

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    AU - Higgins, Denice

    AU - Tobe, Shanan S.

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    AU - Wotherspoon, Andrew

    AU - Gahan, Michelle E.

    AU - McNevin, Dennis

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    N2 - 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.

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