Optimization of DNA isolation and PCR parameters in Myristica sp. and related genera for RAPD and ISSR analysis

Abstract

An efficient protocol for isolation of DNA from wild and related genera of Myristica rich in polysaccharides and polyphenols was developed. The protocol utilizes CTAB (3%), 1.5% PVP and 0.3% B-mercaptoethanol for isolation and RNase and phenol chloroform extraction for purification. The yield of DNA ranged from 25-175 µg/g of fresh leaf tissue, with Knema andamanica giving the highest yield. The present method yielded 10 times higher than the old methods. Characteristic patterns were generated on digestion of DNA by Ecorl and Hind ɪɪɪ restriction enzymes. PCR parameters were optimized using random primers (OPEROTechnology, USA). DNA concentration at 20 ng/reaction, annealing temperature of 45oC, 0.3 mM dNTP in presence of 0.5 U of Taq DNA polymerase, and 2.0 mM MgCl2 and MJ Research Gene Thermocycler was best. Successful amplification by ISSR and RAPD primers indicated that DNA is of good quality and free polysaccharides and polyphenols.