Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry

Thomas A. Hall, Kristin A. Sannes-Lowery, Leslie D. McCurdy, Constance Fisher, Theodore Anderson, Almira Henthorne, Lora Gioeni, Bruce Budowle, Steven A. Hofstadler

Research output: Contribution to journalArticle

37 Scopus citations

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 3 331 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.

Original languageEnglish
Pages (from-to)7515-7526
Number of pages12
JournalAnalytical Chemistry
Volume81
Issue number18
DOIs
StatePublished - 15 Sep 2009

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    Hall, T. A., Sannes-Lowery, K. A., McCurdy, L. D., Fisher, C., Anderson, T., Henthorne, A., Gioeni, L., Budowle, B., & Hofstadler, S. A. (2009). Base composition profiling of human mitochondrial DNA using polymerase chain reaction and direct automated electrospray ionization mass spectrometry. Analytical Chemistry, 81(18), 7515-7526. https://doi.org/10.1021/ac901222y