Rapid and Efficient DNA Extraction Method from Various Plant Species Using Diatomaceous Earth and a Spin Filter
High quality DNA is necessary for genomic library construction, Southern analysis, long-PCR and so on. The CTAB method (Murray and Thompson 1980) is the most popular technique for DNA extraction from plants, but it is time-consuming and is not appropriate for DNA extraction from polysaccharide-or polyphenol-rich plant species. Although a modified method (Wagner et al. 1987) can be used for DNA extraction from such species, it involves numerous additional steps. In addition, in marker-assisted
... n marker-assisted breeding, an easy, rapid and safe DNA extraction method is indispensable. Although many DNA markers associated with agriculturally important traits have been identified, to date there are few cases where DNA marker-assisted breeding has been put into practice. This is because screening based on phenotype is easier, more familiar, less costly and safer than DNA marker detection, which requires DNA extraction. Recently, some simplified protocols have been published (Liu et al. 1995 , Ikeda et al. 2000 . These methods, however, cannot be used for DNA extraction from plant species that have polysaccharide-or polyphenol-rich tissues and hard organs, and in many cases the extracted DNA is not of high quality and is unstable during long-term storage. Here, a method for rapid extraction of high-quality DNA from plants is described. This method was designed based on a plasmid preparation protocol from E. coli that uses diatomaceous earth and a spin filter (Hansen et al. 1995, Kim and Pallaghy 1996) and a method for DNA extraction from plants that does not use phenol or chloroform (Marechal-Drouard and Guillemaut 1995) . This method is as follows. The first step requires grinding of the plant tissue. The second step involves extraction in a buffer containing sodium dodecyl sulfate (SDS) and digestion of RNA by RNase. The third is deproteinization by using potassium acetate. In the fourth step, the DNA is bound to diatomaceous earth in a chaotropic buffer in a spin filter. In the fifth step, the bound DNA is washed with a buffer containing ethanol. Finally, the DNA is eluted from the diatomaceous earth. This method has the following four advantages. (1) The quality of the extracted DNA is high enough for PCR, restriction enzyme digestion, ligation reaction and long-term preservation. (2) The procedure is simple and rapid, because it does not require any centrifugation steps that pellet the DNA. (3) The cost is as low as that of conventional plasmid extraction kits. (4) No dangerous organic solvents such as phenol or chloroform are used. In summary, this method will not only provide a convenient, simple, rapid, low-cost and safe DNA extraction method for molecular biological experiments, but also make possible DNA marker-assisted breeding with rapid and accurate DNA marker detection even in some recalcitrant plant species. Illustrative Protocol Materials and Solutions 1) DNA extraction buffer DNA extraction buffer contains 500 mM Tris-HCl pH 8.0, 50 mM EDTA pH 8.0, 300 mM NaCl, 8 % (v/v) SDS, 1 µg/ml of RNaseA and 0.2 % β-mercaptoethanol (optional). The recipe for 100 ml of DNA extraction buffer is as follows. Prepare sterile stock solutions of 1 M Tris-HCl pH 8.0, 0.5 M EDTA and 5 M NaCl. These can be stored at room temperature after autoclaving. Mix 50 ml of 1 M Tris-HCl pH 8.0 with 10 ml of 0.5 M EDTA, and add 8 g of SDS. After the SDS is dissolved, add 6 ml of 5 M NaCl and mix completely. Bring the total volume to 100 ml with sterilized water, and add 10 µl of 10 mg/ml solution of RNaseA. In order to prevent discoloration caused by oxidation, β-mercaptoethanol can be added just before use. 2) 4 M potassium acetate pH 4.8 Dissolve 23.55 g of potassium acetate in sterilized water to a volume of 66 ml. Add 28.5 ml of glacial acetic acid, mix and titrate with about 1.5 ml of concentrated HCl to a pH of 4.8. Bring the final volume to 100 ml with sterilized water. If necessary, the solution can be sterilized by autoclaving or filter sterilization. Store at room temperature.