Extraction of RAPD-friendly DNA from Laminaria japonica (Phaeophyta) afterenzymatic dissociation of the frozen sporophyte tissues

A new extraction protocol has been developed to obtain high quality DNA fro m Laminaria japonica, which involves enzymatic dissociation of sporophyte t issues and subsequent elimination of the remaining polysaccharides with cet yltrimethyl ammonium bromide. Unicells isolated from frozen kelp tissues wi th alginate lyase prepared from the abalone Haliotis diversicolor were used to extract total DNA; the yield was approximately 13 to 22.5 mug DNA g(-1) (wet sporophyte weight). The average size of genomic DNA was around 23 kb estimated by agarose gel electrophoresis, and the purity of total DNA deter mined spectrophotometrically as the ratio of OD260/OD280 was about 1.7. The extracted kelp DNA (20-40 ng) could be used successfully as a template for polymerase chain reaction (PCR) under the optimized conditions (100 muM dN TP, 0.2 muM primer, 1.0 unit Taq DNA polymerase). The random amplified poly morphic DNA (RAPD) patterns were highly reproducible. These results suggest that the contamination by soluble polysaccharides which interferes with RA PD reproducibility was largely controlled. This RAPD-suited method for DNA extraction from kelp sporophytes using enzyme treatment provided sufficient material, and was inexpensive and convenient to carry out.