| Research Abstract |
In this study, we have developed a new method for species identification by PCR of mtDNA-hypervariable regions. DNA samples were obtained fom blood, hair, and muscle of several animals including human, dog, cat, goat, sheep, ox, rabbit, pig, horse, rat, mouse, ferret, racoon dog, Japnese monkey, gorilla and chimpanzee. Two primer sets were prepared to amplify mtDNA D-loop. One set, mt-U1/mt-U2, amplified the hypervariable region 1 (HV1). The size of PCR products ranged from about 370 by in ferret to 823 by in goat, indicating that animals were distinguishable from each other. Human DNA showed a 545 bp-size, which was similar to the size of ox (543 bp). To dissolve this problem, we prepared the second primer set, mt-HV2F/mt-HV2R, which were specific for human. The HV2/3 product was observed in DNA from human only (600 bp). When duplex PCR was done using the two primer sets, human showed two bands, whereas the other animals, one band. Thus, species was succesfully identified. When unknown samples were not identical in size to DNA samples as a contol, sequencing of PCR poducts and BLAST permitted us to identify species. In fact, when encountering unknown samples in a pratical case, we identified a nucleotide sequence from a weasel (Mustela itatsi) after application of such techniques. In conclusion, since the present species identification method is very simple, easy, rapid, and exact, it is expected to be applied to a field of forensic biology.
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