An Efficient DNA Isolation Method from Nigella sativa L Ranunculaceae Seeds for RAPD and ISSR Analysis

Abstract

Nigella sativa L. (Ranunculaceae) seeds (black cumin) that contain high oil, terpene, protein, alkaloids are an economically important for medicine and food industry. Lack of an efficient DNA isolation procedure is a limiting factor for any molecular studies of these plant. We have used a genomic DNA isolation protocol for Nigella sativa seeds based on a hexadecyltrimethylammonium bromide (CTAB) method described for other plant species. The method is a modified CTAB extraction using high salt concentrations and polyvinyl pyrrolidone (PVP), and successive isoamyl alcohol chloroform extractions and involves mortar grinding of black seeds. Black seeds include little amount of DNA despite high amounts nutrients as polysaccharides and oils. Therefore, the yield was approx., 20 ng DNA per 150 mg of dry seed material. The genomic DNA obtained by this method was suitable to be used in inter-simple sequence repeat (ISSR) and random amplified-polymorphic DNA (RAPD) analyses. This extraction method should facilitate the molecular analysis as genotypic selection via use of dry seeds instead of germination of N. sativa seeds.