TITLE:
Standardization of DNA Extraction Method from Mature Dried Leaves and ISSR-PCR Conditions for Melia dubia Cav. —A Fast Growing Multipurpose Tree Species
AUTHORS:
Swati Rawat, Geeta Joshi, D. Annapurna, A. N. Arunkumar, Nataraja N. Karaba
KEYWORDS:
DNA Extraction, Downstream Applications, ISSR, Mature Dried Leaves, Melia dubia, SSR
JOURNAL NAME:
American Journal of Plant Sciences,
Vol.7 No.3,
March
10,
2016
ABSTRACT: Melia dubia Cav. of family Meliaceae is a fast growing, high value tree species native to India. Isolating DNA from matured dried leaves of M. dubia was difficult due to accumulation of secondary metabolites, majorly polyphenolics, which resulted in dark brown to black colour of the pellet. In this study, a modified STE-(Sucrose, Tris-HCl and Ethylene Diamine Tetra Acetic Acid) CTAB (hexadecyltrimethylammonium bromide) method was standardized for removal of polyphenolics. The protocol developed yielded 200 - 1000 ng/μl of quality DNA without any impurities as evident by A260/280 ratio ranging from 1.75 - 2.0. It was also suitable for extracting quality DNA from other members of Meliaceae like Azadirachta indica and Melia azedarach. In downstream applications, the extracted DNA was used for PCR amplification by using ISSR and SSR markers. ISSR PCR conditions were optimized in a reaction volume of 25 μl, consisting of 30 ng of template DNA, 1.5 mM MgCl2, 200 μM of each of dNTPs and 2 U of Taq polymerase. The best amplification was observed and the same was applicable for SSR markers.