Optimization of DNA isolation and PCR protocol for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India

Abstract

Genetic analysis of plants relies on high yields of pure DNA samples. Here we present the optimization of DNA isolation and PCR conditions for RAPD analysis of selected medicinal and aromatic plants of conservation concern from Peninsular India containing high levels of polysaccharides, polyphenols and secondary metabolites. The method involves a modified CTAB extraction employing polyvinyl pyrrolidone while grinding, successive long-term Chloroform : lsoamyalcohol extractions, an overnight RNase treatment with all steps carried out at room temperature. The yield of DNA ranged from 1-2 μg/μl per gram of the leaf tissue and the purity (ratio) was between 1.6-1.7 indicating minimal levels of contaminating metabolites. The technique is ideal for isolation of DNA from different plant species and the DNA isolated was used for randomly amplified polymorphic DNA (RAPD) analysis. RAPD protocol was optimized based on the use of higher concentration of MgCl₂ (3 mM), lower concentrations of primer (0.5 μM) and Taq polymerase (0.2 units), 50 ng of template DNA and an annealing temperature of 37°C, resulted optimal amplification. Reproducible amplifiable products were observed in all PCR reactions. Thus the results indicate that the optimized protocol for DNA isolation and PCR was amenable to plant species belonging to different genera which is suitable for further work on diversity analysis.

Keywords: Vitex pubescens, Nervilia aragoana, Gymnema sylvestre, Withania somnifera, Origanum majorana, Boswellia serrata, Saraca asoca, Gloriosa superba, polysaccharides, PCR amplification