Accurate determination of allelic frequencies in mitochondrial DNA mixtures by electrospray ionization time-of-flight mass spectrometry.

The mitochondrial locus 16519T/C was used as a model for the evaluation of the benefits of ion-pair reversed-phase high-performance liquid chromatography on-line hyphenated to electrospray ionization time-of-flight mass spectrometry (ICEMS assay) for the determination of allelic frequencies of single nucleotide polymorphisms. This marker has gained interest in forensic science owing to its ability to increase the discrimination power of mitochondrial DNA testing as a consequence of its high variability across various populations. In a first set of experiments, artificial mitochondrial DNA mixtures prepared from all four theoretically possible 16519 alleles served as samples. Any allele occurring at a frequency of as low as 1-5% was unequivocally detectable irrespective of the kind of allelic mixture. Measured and expected allelic frequencies correlated well following correction of observed experimental bias, which was most probably attributable to differential PCR amplification and/or preferential ionization. For thirteen different T/C mixtures with C contents in the range 1.0-99.0%, an average error of 1.2% and a maximum error of 2.2% were observed. Furthermore, ICEMS was applied to the quantitative genotyping of eight selected individuals of which four were heteroplasmic with C contents in the range 1.9-34.1%. To check the reliability of these results, allelic proportions were additionally determined by a cloning assay. The results of the two assays correlated well (R (2)=0.9971). In all cases, deviations were obtained that were smaller than 5.4%. The overall observed assay performance suggests that the described mass spectrometric technique represents one of the most powerful assays for the determination of allelic frequencies available today.