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 journalArticle

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

Fingerprint Dive into the research topics of 'Internal validation of human mitochondrial DNA quantification using real-time PCR'. Together they form a unique fingerprint.

  • Cite this