Sources of DNA and amplification of COI gene of Changeable Hawk-eagle Nisaetus cirrhatus (J.F. Gmelin 1788)

The mitochondrial gene of cytochrome c oxidase I (COI) has been commonly used in population genetics, systematics, phylogeography as well as DNA barcoding of many animal species including birds. This study aimed to evaluate the sources of DNA and condition of PCR reaction for amplification of COI gene of changeable hawk-eagle (Nisaetus cirrhatus Gmelin, JF, 1788). DNA was extracted from single fresh plucked feather and from cloacal swab of five individuals. The DNA extraction was conducted using Geneaid™ DNA Isolation Kit (Tissue). Amplification of COI gene was done in a reaction mixture containing 1x PCR buffer, 0.2mM dNTP, 2mM to 3mM MgCl2, 1μM of each forward and reverse primers, 1μl to 3.5μl DNA template, 1U taq polymerase and H2O to reach a total volume of 20μl The PCR cycles were 95°C for 1 minute followed by 5 cycles of 95°C for 1 minute, 45°C for 1.5 minutes, and 72°C for 1.5 minutes. This was followed by 30 cycles of 1 minute at 95°C, 1.5 minutes at 50°C, and 1.5 minute at 72°C. One cycle of a final extension was done for 5 minutes at 72°C. The amplification products were visualised using agarose gel electrophoresis. The results showed that DNA extracted from feather had higher concentration than from cloacal swab. Moreover, DNA extracted from plucked feather demonstrated better amplification product with the fragment size of 750bp. The volume of DNA template from plucked feather that resulted in reproducible amplification product was 3.5 μl and the MgCl2 concentration was 3mM.