Optimization of DNA isolation for RAPD-PCR analysis of selected (Echinaceae purpurea L. Moench) medicinal plants of conservation concern from Turkey.

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 plants of conservation concern from Turkey, containing high levels of polysaccharides, polyphenols and secondary metabolites. The method involves a modified CTAB extraction employing polyvinyl pyrrolidone (PVP) while grinding, successive long-term chloroform-isoamylalcohol extractions, EZ1 nucleic acid isolation protocols. The yield of DNA ranged from 1 - 2 μg/μl per gram of the leaf tissue and the purity (ratio) was between 1.7 - 1.8 indicating minimal levels of contaminating metabolites. EZ1 nucleic acid isolation 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. Furthermore, here we used suitable DNA isolation protocol for RAPD analysis to study the genetic variation in the future in Echinaceae sp. grown in Turkey. Key words: Echinaceae purpurea, random amplified polymorphic DNA, polymerase chain reaction, medicinal plant.