Internal validation of human mitochondrial DNA quantification using real-time PCR

Marc L. Sprouse, Nicole R. Phillips, Mark F. Kavlick, Rhonda K. Roby

Research output: Contribution to journalArticlepeer-review

11 Scopus citations

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.

Original languageEnglish
Pages (from-to)1049-1056
Number of pages8
JournalJournal of Forensic Sciences
Volume59
Issue number4
DOIs
StatePublished - Jul 2014

Keywords

  • Forensic science
  • Inhibition studies
  • Internal validation
  • Mitochondrial DNA
  • Quantitative PCR
  • Sequence analysis

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