Isolation of milligram quantities of nuclear DNA from tomato (Lycopersicon esculentum), A plant containing high levels of polyphenolic compounds
We have developed a protocol for isolating milligram quantities of highly purified DNA from tomato nuclei. The protocol utilizes fresh seedlings or leaves without freezing. Tissues are treated with ethyl ether, thoroughly washed, and placed in a buffer containing the nuclear-stabilizing agent 2-methyl-1,4-pentanediol. Nuclei are liberated from tomato cells by homogenization in a Waring blender. The interaction of nuclear DNA with oxidized polyphenols is inhibited by compounds that adsorb polyphenols or prevent oxidation reactions. Chloroplasts and mitochondria are preferentially eliminated with Triton X-100. Nuclei are concentrated using a Percoll gradient and lysed with SDS. DNA is subsequently purified by RNase and protease digestions and phenol/chloroform extractions. The isolated DNA is essentially free of polyphenols and other major contaminants based upon its lack of coloration, A260/A280 ratio, digestibility with restriction enzymes, melting profile, and reassociation properties.
Keywordstomato Lycopersicon esculentum polyphenols tannins DNA nuclei
Unable to display preview. Download preview PDF.
- Couch, J.A. and P.J. Fritz. 1990. Isolation of DNA from plants high in polyphenolics. Plant Mol. Biol. Reptr. 8:8–12.Google Scholar
- Guillemaut, P. and L. Maréchal-Drouard. 1992. Isolation of plant DNA: A fast, inexpensive, and reliable method. Plant Mol. Biol. Reptr. 10:60–65.Google Scholar
- Loomis, W.D. 1974. Overcoming problems of phenolics and quinones in the isolation of plant enzymes and organelles. Meth. Enzymology 31:528–544.Google Scholar
- Peterson, D.G. and S.M. Stack. 1997a. A method for isolating milligram quantities of “polyphenol-free” nuclear DNA from tomato. Report Tomato Genet. Coop. 46:in press.Google Scholar
- Peterson, D.G. and S.M. Stack. 1997b. Characterization of the tomato genome using Cot analysis. Report Tomato Genet. Coop. 46:in press.Google Scholar
- Peterson, D.G. and S.M. Stack. 1997c. GC content for tomato DNA based on DNA melting. Report Tomato Genet. Coop. 46in press.Google Scholar
- Sambrook, J., E.F. Fritsch and T. Maniatis. 1989.Molecular Cloning: A Laboratory Manual, 2nd edition, C. Nolan, ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.Google Scholar