title: Optimization of human mtDNA control region sequencing for forensic applications.
abstract: Sequencing mitochondrial DNA hypervariable regions I and II (HVI and HVII) is useful in forensic missing person and unidentified remains cases. Improvements in ease and sensitivity of testing will yield results from more samples in a timely fashion. Routinely, amplification of HVI and HVII is followed by Sanger sequencing using the BigDye(®) Terminator v3.1 Cycle Sequencing kit (Applied Biosystems) using 4 μL of ready reaction mix (RRM). Each sequencing reaction is then purified through column filtration before capillary electrophoresis. Using lower amounts of RRM (2 μL or 1 μL) and purification using BigDye(®) XTerminator(™) (Applied Biosystems) instead of columns showed no loss of sequence length and increased the quality and the sensitivity of testing, allowing HVI and HVII typing from mitochondrial genome equivalent to 125 fg of nuclear DNA, or 100 pg of HVI/HVII amplicons. Using this methodology, testing can be completed in 1 day, and the cost of testing is reduced.
authors.affiliation: Department of Forensic Biology, The City of New York Office of Chief Medical Examiner, 421 East 26th Street, New York, NY.
authors.forename: Véronique
authors.initials: V
authors.lastname: Bourdon
authors.forename: Carolyn
authors.initials: C
authors.lastname: Ng
authors.forename: Jessica
authors.initials: J
authors.lastname: Harris
authors.forename: Mechthild
authors.initials: M
authors.lastname: Prinz
authors.forename: Eli
authors.initials: E
authors.lastname: Shapiro
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial
keywords: forensic science; hair; hypervariable regions I and II; mitochondrial DNA; optimization; sequencing