Internal validation of human mitochondrial DNA quantification using real-time PCR.
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.
Department of Molecular and Medical Genetics, University of North Texas Health Science Center, Fort Worth, TX, 76107.
Marc L
ML
Sprouse
Nicole R
NR
Phillips
Mark F
MF
Kavlick
Rhonda K
RK
Roby
D016172:DNA Fingerprinting; D004272:DNA, Mitochondrial / Q000032:analysis*; D006801:Humans; D016014:Linear Models; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods
D004272:DNA, Mitochondrial
forensic science; inhibition studies; internal validation; mitochondrial DNA; quantitative PCR; sequence analysis