Using species-specific repeat and PCR-RFLP in typing of DNA derived from blood of human and animal species.

Species determination of tissue specimens, including blood, is an important component of forensic analysis to distinguish human from animal remains. DNA markers based on a method of species-specific PCR and amplifying the 359-base pair (bp) fragment of the mitochondrially encoded cytochrome-b gene and then digestion with the TaqI restriction enzyme were developed for detection and discrimination of human, cattle, buffalo, horse, sheep, pig, dog, cat and chicken blood samples. The results reveal that PCR-amplification of the gene encoding the species-specific repeat (SSR) region generated 603 bp in cattle and buffalo, 221 bp in horse, 374 bp in sheep, <or=100 bp in pig, 808 bp in dog, 672 bp in cat and 50 bp in chicken. Restriction analysis of the amplified 359-bp portion of the cytochrome-b gene using the TaqI restriction enzyme results in species-specific restriction fragment length polymorphism (RFLP) between buffalo, cattle and human. Two different bands were generated in buffalo (191 and 168 bp) and human (209 and 150 bp), with no digestion in cattle (359 bp). Cytochrome-b is a highly conserved region and consequently a good molecular marker for diagnostic studies. Therefore, the two complementary techniques, SSR-PCR and PCR-RFLP, could be used successfully as routine methods in forensics for sensitive, rapid, simple and inexpensive identification of the species in bloodstains.