Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components
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A relatively quick, inexpensive and consistent protocol for extraction of DNA from expanded leaf material containing large quantities of polyphenols, tannins and polysaccharides is described. Mature strawberry leaves, which contain high levels of these secondary components, were used as a study group. The method involves a modified CTAB extraction, employing high salt concentrations to remove polysaccharides, the use of polyvinyl pyrrolidone (PVP) to remove polyphenols, an extended RNase treatment and a phenol-chloroform extraction. Average yields range from 20 to 84 μg/g mature leaf tissue for both wild and cultivated octoploid and diploidFragaria species. Results from 60 plants were examined, and were consistently amplifiable in the RAPD reaction with as little as 0.5 ng DNA per 25-μL reaction. Presently, this is the first procedure for the isolation of DNA from mature strawberry leaf tissue that produces consistent results for a variety of different species, both octoploid and diploid, and is both stable and PCR amplifiable before and after extended storage. This procedure may prove useful for other difficult species in the family Rosaceae.
Key WordsFragaria DNA extraction RAPD strawberry
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- Collins, G.G. and R.H. Symons. 1992. Extraction of nuclear DNA from grape vine leaves by a modified procedure. Plant Mol. Biol. Rept. 10:233–235.Google Scholar
- Dellaporta, S.L., J. Wood and J.B. Hicks. 1985. Maize DNA miniprep, p. 36–37, inMolecular Biology of Plants, Malberg, J. Messing and I. Sussex, eds Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
- Doyle J.J. and J.L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bull 19:11–15.Google Scholar
- Fang, G., S. Hammar and R. Grumet. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Biofeedback 13 (1):52–54.Google Scholar
- LaRoche, J. 1992. An easy and efficient procedure for isolating plant DNA using the Elu-Quik DNA purification kit. Sequences 36:3–4.Google Scholar
- Lodhi, M.A., G.-N. Ye, N.F. Weeden, B.I. Reisch. 1994. A simple and efficient method for DNA extractiosn from grapevine cultivars and Vitis species. Plant Mol. Biol. Rept. 12:6–13.Google Scholar
- Mauro, M.-C., M. Strefeler, N.F. Weeden and B.I. Reisch. 1992. Genetic analysis of restriction fragment length polymorphisms inVitis. J. Heredity 83:18–21.Google Scholar
- Oard, J.H. and S. Dronavalli. 1992. Rapid isolation of rice and maize DNA for analysis by random-primer PCR. Plant Mol. Biol. Rept. 10:236–241.Google Scholar
- Richards E., M. Reichardt and S. Rogers. 1994. Preparation of genomic DNA from plant tissue.Current Protocols in Molecular Biology. Wiley Interscience.Google Scholar
- Sigma Chemical Company. 1993.Biochemical Organic Compounds for Research and Diagnostic Reagents. Sigma Diagnostics. St. Louis, USA. 2256 pp.Google Scholar
- Varadarajan, G.S. and C.S. Prakash. 1991. A rapid and efficient method for the extraction of total DNA from the sweet potato and its related species. Plant Mol. Biol. Rept. 9(1):6–12.Google Scholar