An efficient genomic DNA isolation protocol for RAPD and SSR analysis in Acorus calamus L.

摘要

Molecular based genetic analysis requires good quality of DNA in sufficient quantity to generate robust DNA fingerprints. The leaves of sweet flag (Acorus calamus L.) contain high amounts of polyphenolic compounds and polysaccharides, which interfere in the amplification reactions. Four genomic DNA extraction methods were tested for yield, quality and suitability of genomic DNA for RAPD (random amplified polymorphic DNA) and SSR (simple sequence repeat) marker analysis in sweet flag. Fresh young leaves were subjected to the already available protocols and a procedure was devised after modification in the protocol of Stange et al for the isolation of total genomic DNA. The modified method employs high concentrations of polyvinyl pyrrolidone, addition of lithium chloride solution as well as additional washing step of DNA pellets. The yield was found approximately 200-500 mu g DNA per 100 mg of plant material and the purity ratio was found 1.7-2.0. Usage of highly concentrated PVP extraction buffer and chloroform:isoamylalcohol repetition step was found useful to overcome problems from polyphenolic compounds and polysaccharides. The extracted DNA through this method was found suitable for RAPD and SSR analysis.