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Internal Validation of Human Mitochondrial DNA Quantification Using Real‐Time PCR
Author(s) -
Sprouse Marc L.,
Phillips Nicole R.,
Kavlick Mark F.,
Roby Rhonda K.
Publication year - 2014
Publication title -
journal of forensic sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.715
H-Index - 96
eISSN - 1556-4029
pISSN - 0022-1198
DOI - 10.1111/1556-4029.12477
Subject(s) - mitochondrial dna , dna , microbiology and biotechnology , polymerase chain reaction , dna sequencing , real time polymerase chain reaction , biology , reproducibility , terminator (solar) , human mitochondrial genetics , chromatography , computational biology , chemistry , genetics , gene , ionosphere , physics , astronomy
The quantity of mitochondrial DNA (mt DNA ) template added for amplification and subsequent dye terminator reactions is critical for obtaining quality sequence data. Validation of a human mt DNA 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 mt DNA template is recommended for successful HV 1 and HV 2 sequence analysis; however, as little as 0.013 pg can generate a full mt DNA 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 mt DNA and is useful for determining the best analytical approach for successful sequencing.