A Simple and Effective Phenol-Chloroform Method of DNA Extraction from Mammalian Feces

Aims: To non-invasively collect the fecal samples of 12 different mammals and devise a simple method of fecal DNA extraction using a modified phenol-chloroform procedure of DNA isolation for species identification.

Study Design: The experiment was laid out to check the applicability of the devised protocol. Morphological identification was done in the field to collect samples from the wild. Molecular characterization was carried out in the Molecular laboratory for species identification.

Place and Duration of Study: Advanced Institute for Wildlife Conservation (AIWC), Tamil Nadu Forest Department, Vandalur, Chennai, Tamil Nadu. The samples were collected between the time period of September 2019 and March 2020. The molecular analysis was performed between July 2020 and July 2021.

Methodology: We devised a scat/ fecal DNA isolation protocol and tested its applicability on 81 samples (30 herbivores, 15 omnivores, 36 Carnivores). Fresh and old samples were collected from wild (n=41) and captive (n=40) areas and used for the study. Independent isolation for each species was carried out with extraction control. The DNA isolated samples were quantified for concentration using Nanodrop Spectrophotometer to calculate the optimum DNA concentration for amplification. Independent PCR amplification of mitochondrial regions of cytochrome b and 12S rRNA were performed and gel electrophoresis was carried out for each sample for positive amplicons. The PCR products were sequenced using genetic analyzer.

Results: The protocol was validated by checking the strength of the devised method to work on species belonging to different ecological types. The sample size is n=81, positive amplification in cytochrome b region is 71 and 12S rRNA region is 79. Success of devised DNA extraction protocol on different population kinds, such as wild (n=41) and captive (n=40) were evaluated with a ratio of ‘Positive PCRs of samples’ against ‘Total samples for PCR’. It is 97.56 % (cytochrome b) and 100 % (12S rRNA) of Wild population and 60.0 % (cytochrome b) and 95% (12S rRNA) of captive population.

Conclusion: The devised protocol successfully worked on both wild and captive populations of herbivores, omnivores and carnivores. The success rate is better in 12S rRNA region on comparison with Cytochrome b. The applicability and reliability of the protocol has been tested and validated by checking the obtained sequences in the NCBI database and submitting the same to the database.