OPTIMIZATION OF DNA EXTRACTION AND PCR PROTOCOL FOR RAPD ANALYSIS OF TOR PUTITORA - N. Shafi; S. Janjua; S. Jafar K. Anwar; A. Mian (Journal of Animal and Plant Sciences, 2015)
Genetic analysis of biological organisms is dependent on a high yield of genomic DNA samples. This study was designed for the optimization of DNA extraction and PCR protocol for RAPD analysis of golden mahseer (Pisces: Tor putitora) to find out the genetic diversity among its different populations. The extraction is a modified phenol-chloroform method involving addition of 40 µL of proteinase K during overnight lysis of samples and successive long-term chloroform- isoamylalcohol extractions. The yielded DNA showed clear thick bands on agarose gel and its quality was not affected by the period of preservation. The isolated DNA was used for randomly amplified polymorphic DNA (RAPD) analysis. The protocol was optimized based on higher concentration of MgCl (2.5 mM), lower concentrations of primers (0.25 ρmol) 2 and Taq polymerase (0.02 units), 50 ng of template DNA and an annealing temperature of 34°C. Thus on the basis of results this protocol for DNA isolation and PCR was accurate for the genetic diversity analysis of different populations of Masheer. sequencing or any other form of the molecular
Keywords: journal article; Mahseer; Tor putitora; DNA extraction; PCR optimization; reaction parameters