title: Quantification of human mitochondrial DNA using synthesized DNA standards.
abstract: 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.
authors: Counterterrorism and Forensic Science Research Unit, Laboratory Division, Federal Bureau of Investigation, 2501 Investigation Parkway, Quantico, VA 22135, USA.
authors: Mark F
authors: MF
authors: Kavlick
authors: Helen S
authors: HS
authors: Lawrence
authors: R Travis
authors: RT
authors: Merritt
authors: Constance
authors: C
authors: Fisher
authors: Alice
authors: A
authors: Isenberg
authors: James M
authors: JM
authors: Robertson
authors: Bruce
authors: B
authors: Budowle
mesh_terms: D000818:Animals; D022801:Complementarity Determining Regions / Q000235:genetics; D016172:DNA Fingerprinting / Q000592:standards*; D017931:DNA Primers; D015342:DNA Probes; D004272:DNA, Mitochondrial / Q000032:analysis*; D006801:Humans; D016014:Linear Models; D011786:Quality Control; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D022801:Complementarity Determining Regions; D017931:DNA Primers; D015342:DNA Probes; D004272:DNA, Mitochondrial