A technique for the quantification of human and non-human mammalian mitochondrial DNA copy number in forensic and other mixtures

The number of mitochondria per cell varies by cell type and the number of mitochondrial DNA (mtDNA) genomes varies per mitochondrion. Biological samples from unknown species are encountered frequently in forensic science investigations and are often contaminated with human mtDNA making analysis difficult. Currently, no techniques to quantify non-human mtDNA are available. We report on a method to accurately quantify, sensitive to 100 copies (1.7 fg), mtDNA from human and non-human sources when present as a mixture. The test developed uses the cytochrome b (cytb) and the ribosomal 12S genes on the mitochondrial genome. Universal and human specific fragments of similar size are amplified and quantified using SYBR Green. We validate the test with 24 human samples and 27 non-human mammalian samples. The human fraction of a sample can then be subtracted from the universal fraction for an accurate estimation of non-human mtDNA copy number.