One of the objectives of the ongoing research project is to identify the species from bones, horns, antlers and tusks through DNA based techniques. We standardized protocols at different stages, mainly for extraction and amplification of DNA. Different extraction protocols were required to be applied with bones, horns, antlers and tusks and similarly standardization for PCR amplifications. We tried three different methods for DNA extraction from bones (n=10) viz, three protocols of Gene Clean® (Bio 101 systems, Q Biogene, USA), Bio-Robot EZ1® (Qiagen, Germany) and Phenol Chloroform. The extraction protocols were followed as recommended, but the results were not satisfactory .Most of the time the DNA yield was low with small fragment <300bp and inhibitors were present. Thus we modified Gene Clean® protocol and good quality DNA having up to 600bp was obtained. This has allowed successful amplification of cyt b region of 450 bp. The PCR product was sequenced. We also standardized Bio-Robot EZ1® protocols for bones and much better yield and DNA up to 900 bp was obtained. We feel that there is a need to have further modification which will improve the results by using centricon columns and with gel purification (Qia Quick Gel Extraction Kit, Qiagen Germany) for removal of inhibitors. Attempts were also made to extract DNA from rhino and buffalo horns using Qiagen tissue kit (Qiagen Germany) which has yielded up to 200 bp DNA. Modification in this method by using dithiothritol has improved DNA yield. DNA of < 200 bp was obtained in case of antler powder using Gene Clean® protocol. Published protocols for elephant tusk have been tried and work is in progress.

Dr. S.P. Goyal.