Internal Validation of Human Mitochondrial DNA Quantification Using Real-Time PCR

Authors

  • Marc L. Sprouse B.S.,

    1. Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX
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  • Nicole R. Phillips Ph.D.,

    1. Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX
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  • Mark F. Kavlick B.S.,

    1. Counterterrorism and Forensic Science Research Unit, Laboratory Division, Federal Bureau of Investigation, Quantico, VA
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  • Rhonda K. Roby Ph.D., M.P.H.

    Corresponding author
    1. Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX
    2. Institute of Applied Genetics, University of North Texas Health Science Center, Fort Worth
    • Additional information and reprints requests:

      Rhonda K. Roby, Ph.D., M.P.H.

      UNT Health Science Center

      Department of Molecular and Medical Genetics

      3500 Camp Bowie Boulevard, CBH-363

      Fort Worth, TX 76107

      E-mail: Rhonda.Roby@unthsc.edu

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  • Presented at the 64th Annual Meeting of the American Academy of Forensic Sciences, February 20–25, 2012. Support provided by the National Institute of Justice, Forensic DNA Unit Efficiency Improvement Program (2008-DN-BX-K192 and 2009-DN-BX-K171) and the National Institute on Aging, Training in the Neurobiology of Aging (T32-AG-020494).

Abstract

The quantity of mitochondrial DNA (mtDNA) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mtDNA real-time quantitative PCR (qPCR) assay demonstrated its high degree of reproducibility and precision as well as an extremely sensitive threshold of detection (0.0001 pg/μL or approximately six human mtDNA copies/μL). A study of 35 nonprobative bone and teeth evidence samples revealed that 20 pg of mtDNA template is recommended for successful HV1 and HV2 sequence analysis; however, as little as 0.013 pg can generate a full mtDNA profile when using enhanced amplification reactions. The assay can also detect PCR inhibition and is useful for identifying samples that may benefit from re-purification. Overall, the assay is an excellent method to quantify mtDNA and is useful for determining the best analytical approach for successful sequencing.

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