Quantification of Human Mitochondrial DNA Using Synthesized DNA Standards

• Published in: Journal of forensic sciences 2011-11, Vol.56 (6), p.1457-1463
• Main Authors: Kavlick, Mark F., Lawrence, Helen S., Merritt, R. Travis, Fisher, Constance, Isenberg, Alice, Robertson, James M., Budowle, Bruce
• Format: Article
• Language: English
• Subjects: Amplification; Animals; assay; Assaying; Complementarity Determining Regions - genetics; Deoxyribonucleic acid; DNA; DNA Fingerprinting - standards; DNA Primers; DNA Probes; DNA quality; DNA quantification; DNA, Mitochondrial - analysis; forensic science; Forensic sciences; Gene amplification; Homology; Humans; Linear Models; Mitochondrial DNA; Polymerase chain reaction; Polymorphism; Quality assurance; Quality Control; quantitative PCR; Real-Time Polymerase Chain Reaction; Reproducibility of Results; Sequence Analysis, DNA; Species Specificity; synthetic DNA standards
• ISSN: 0022-1198; 1556-4029
• DOI: 10.1111/j.1556-4029.2011.01871.x

:  Successful mitochondrial DNA (mtDNA) forensic analysis depends on sufficient quantity and quality of mtDNA. A real‐time quantitative PCR assay was developed to assess such characteristics in a DNA sample, which utilizes a duplex, synthetic DNA to ensure optimal quality assurance and quality control. The assay’s 105‐base pair target sequence facilitates amplification of degraded DNA and is minimally homologous to nonhuman mtDNA. The primers and probe hybridize to a region that has relatively few sequence polymorphisms. The assay can also identify the presence of PCR inhibitors and thus indicate the need for sample repurification. The results show that the assay provides information down to 10 copies and provides a dynamic range spanning seven orders of magnitude. Additional experiments demonstrated that as few as 300 mtDNA copies resulted in successful hypervariable region amplification, information that permits sample conservation and optimized downstream PCR testing. The assay described is rapid, reliable, and robust.