Revista Europea de Biología Experimental Acceso abierto

Abstracto

Optimization of DNA isolation and RAPD-PCR protocol of Acanthus volubilis wall., a rare mangrove plant from Indian Sundarban, for conservation concern

Surya Shekhar Das, Swati Das (Sur) and Parthadeb Ghosh

Extraction of pure and high molecular weight genomic DNA is a prerequisite for molecular biological studies of an organism. However, the presence of polysaccharides and polyphenols in plants upset the isolation of pure DNA and downstream reactions like PCR amplification. Here we present the optimization of DNA isolation protocol and PCR conditions for RAPD analysis of Acanthus volubilis, as available standard protocols do not produce high quality PCR amplifiable DNA. The method involves a modified CTAB extraction employing 2M NaCl, 2.5% CTAB, 3.0 % β- mercaptoethanol, 4.0% PVP and 0.13% sodium sulphite. The yield of DNA was 82.14 μg per gram of leaf tissue and the A260/A280 value was 1.79 indicating minimal levels of contamination. RAPD protocol was optimized based on the use of 3mM MgCl2, 200 μM dNTP mix, 0.2 unit of Taq DNA polymerase, 5 picomoles of single random decamer primer and 25ng of template DNA. An annealing temperature of 380C resulted in optimal amplification. In all PCR reactions reproducible amplified products were observed. High intensity amplification with random decamer primers during PCR also indicates that the DNA was of good quality and free from interfering compounds. Thus the results indicate that the optimized protocol for DNA isolation and RAPD-PCR will aid in further work on genetic diversity analysis, phylogenetic studies and most importantly in developing conservation strategies of this very rare mangrove plant from Indian Sundarban