While dealing with wildlife offence cases at WII, hair samples, tanned & semi-tanned skins samples have been most frequently encountered as physical evidence. In certain cases the hair samples and small skin pieces may not be enough to make conclusive opinion by microscopic examination. Attempts were made to standardize protocols for extraction and amplification of DNA from hair, tanned & semi-tanned skin samples. Hair samples obtained as physical evidence may be with or without root & follicular tissue. Extraction of DNA from such exhibits becomes a difficult task due to limitation on sample size, quality and fear of losing the trace evidence in the process. We tried six different type of extraction protocols viz. Qiagen ® (Qiagen, Germany), Bio-robot EZ1® (Qiagen, Germany), phenol chloroform, Gene Clean® (Q Biogene,, USA) , Chelex and alkali digestion to extract DNA from the above mentioned two types of hair samples and the first four protocols were used for skin (tanned & semi-tanned) samples. The PCR amplification for the extracted DNA was checked with primers targeting different regions of mitochondrial DNA i.e cytochrome b (480bp) and 16s r RNA (540 bp). Sequencing was further done for the amplified fragments to generate database of sequences for protected species. The extracted DNA in most cases (85% samples) could not be visualized on agarose gel due to low concentration. PCR amplification success rates for mitochondrial DNA were checked with various primers. In case of hair samples, Qiagen and Robotic extraction were found to be the best protocols where PCR inhibition was the minimum and all samples (n=25) amplified. DNA extraction was possible with all protocols in case of semi-tanned skins and mitochondrial cytochrome b (480bp) and 16s r RNA (540 bp) fragments could be amplified (85% success). In case of tanned skins Robotic extraction and Gene Clean were found to be most efficient where 381 bp fragment of mitochondrial cytochrome b gene was successfully amplified. Primers amplifying more than 400 base pairs did not yield any positive results in tanned skins. Based on the above work 10 sequences were submitted to GenBank (National Center for Biotechnology Information, USA) for dusky musk deer (Moschus fuscus), Serow (Capricornis sumtraensis), mouse deer (Tragulus memmina), chinkara (Gazella bennettii) and black buck (Antilope cervicapra).

Dr. S.P. Goyal.