Identification of human mitochondrial DNA based on amplification of human-specific mitochondrial sequence and its forensic application

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Species-specific differences in a non-polymorphic region of the mitochondrial genome appear to be large enough to allow human-specific amplification of forensic DNA samples. On the other hand, sequence variation within the human mtDNA control region (hypervariable D-loop) has been widely established as a valuable marker for personal identification. We therefore developed a method of multiplex-PCR amplification of both NADH dehydrogenase subunit 4(ND4) and hypervariable D-loop region allowing species determination and personal identification. Following the multiplex-PCR amplification, amplicons are first screened on an agarose gel. The presence of double-bands indicates that the sample is human origin, while the absence of ND4 fragment indicates a non-human. Subsequent DNA sequencing of the hypervariable D-loop region allows for personal identification as the presence of ND4 fragment dose not interfere with the analysis. In addition, species and individual identification were successfully made of aged biological specimens comprising bloodstains, hair shafts and bones, illustrating the applicability of the present method to forensic specimens.