Validation of mass spectrometry analysis of mitochondrial DNA

The analysis of mitochondrial DNA (mtDNA) is used routinely to assist in determining the source of old bones, teeth, hair shafts, and other challenged biological samples. Sequencing is the method of choice for typing mtDNA. However, the method is laborious, time consuming, costly and currently not a practical approach for typing single nucleotide polymorphisms (SNPs) contained within the coding region of the mtDNA genome. Electrospray ionization mass spectrometry (ESI-MS) has several advantages for mtDNA analysis including speed, sensitivity and the ability to interrogate mixtures. As with sequencing and unlike many other SNP-based approaches, individual polymorphic positions do not need to be specifically targeted, as all variation within amplified regions is assayed simultaneously. Since each amplicon is assayed as an individual component, the technique easily resolves length heteroplasmy and is capable of quantitatively analyzing heteroplasmic and mixed samples. The ESI-MS approach originally described for mtDNA analysis has been improved using the Ibis T5000™ and a 24 amplicon tiling format and validation studies are underway. Results from these studies support that the use of mass spectrometry is a robust and reliable method for mtDNA analysis that offers resolution approaching that of sequencing, the ability to deconvolve mixtures and analyze heteroplasmy effectively, and a sensitivity of detection equivalent to current methods, with at least an order of magnitude increased throughput and a reduction in cost.