title: Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry.
abstract: We describe an automated system for high-resolution profiling of human mitochondrial DNA (mtDNA) based upon multiplexed polymerase chain reaction (PCR) followed by desolvation and direct analysis using electrospray ionization mass spectrometry (PCR/ESI-MS). The assay utilizes 24 primer pairs that amplify targets in the mtDNA control region, including the hypervariable regions typically sequenced in a forensic analysis. Profiles consisting of product base compositions can be stored in a database, compared to each other, and compared to sequencing results. Approximately 94% of discriminating information obtained by sequencing is retained with this technique. The assay is more discriminating than sequencing minimum HV1 and HV2 regions because it interrogates more of the mitochondrial genome. A profile compared to a population database can be subjected to the same statistics used for assessing the significance of concordant mtDNA sequences. The assay is not hindered by length heteroplasmy, can directly analyze template mixtures, and has a sensitivity of <25 pg of total DNA per reaction. Analysis of 3331 independent trials of the same sample over 28 months produced an average mass measurement uncertainty of 10.1 +/- 8.0 ppm, with >99% of trials producing a full profile with automated analysis. The technique has direct application to analysis of forensic biological evidence.
authors.affiliation: Ibis Biosciences, Abbott Molecular, Inc., Carlsbad, California 92008, USA.
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Hall
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Sannes-Lowery
authors.forename: Leslie D
authors.initials: LD
authors.lastname: McCurdy
authors.forename: Constance
authors.initials: C
authors.lastname: Fisher
authors.forename: Theodore
authors.initials: T
authors.lastname: Anderson
authors.forename: Almira
authors.initials: A
authors.lastname: Henthorne
authors.forename: Lora
authors.initials: L
authors.lastname: Gioeni
authors.forename: Bruce
authors.initials: B
authors.lastname: Budowle
authors.forename: Steven A
authors.initials: SA
authors.lastname: Hofstadler
mesh_terms: D001482:Base Composition; D004272:DNA, Mitochondrial / Q000737:chemistry*; D030541:Databases, Genetic; D053803:Forensic Genetics; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004272:DNA, Mitochondrial