An effective method for extraction and polymerase chain reaction (PCR) amplification of DNA from formalin preserved tissue samples of snow leopard

Abstract

Formalin-preserved biological samples obtained from endangered species are valuable in assessing genetic diversity. To make use of snow leopard samples preserved in formalin over a period of two to seven years, we optimized the method of extracting DNA from these samples. We used (a) phenol chloroform : isoamyl alcohol, (b) the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Germany), (c) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with NaOH for three days and (d) the Qiagen DNeasy Blood and Tissue Kit after treating the samples with phosphate buffered saline (PBS) for three days. The usefulness of the extracted DNA was assessed on the basis of mitochondrial (150 to 550 bp) and nuclear (95 to 229 bp) markers. There was no PCR amplification with the first two methods. The PCR amplification with the NaOH and PBS treatment had a success rate of 30 to 100% for both mitochondrial and nuclear markers. The PBS method is the best method for extraction of DNA from formalin-preserved samples of longer period (two to seven years) because of higher success rate in amplifying mitochondrial gene of ca. 550 bp (60%) than the NaOH method (28%). The overall amplification of microsatellite markers in such samples was also higher in samples treated with PBS (43 to 100%) than NaOH (0 to 100%). The PCR products obtained were confirmed through DNA sequencing to be of snow leopard origin. The optimized protocol will enable genetic studies to be conducted on tissue samples of other species that have been preserved in formalin. The protocol will be particularly useful for species that are elusive and from which it is difficult to collect fresh tissue samples.

Keywords: Formalin, polymerase chain reaction (PCR), mtDNA, microsatellites, snow leopard

African Journal of Biotechnology Vol. 12(22), pp. 3399-3404