Advertisement

Chloroplast DNA Isolation

  • George S. Mourad
Part of the Methods in Molecular Biology™ book series (MIMB, volume 82)

Abstract

The size of the chloroplast DNA molecule of Arabidopsis thaliana has been determined to be about 153 kb (1). In this chapter, the goal is to provide a step—by—step laboratory procedure for isolating chloroplast DNA from Arabidopsis thaliana with minimal nuclear or mitochondrial DNA contamination. In general, a first step in isolating chloroplast DNA is the homogenization of the plant material followed by a filtration step to remove large-sized cell debris and cell fragments. The filtrate is then centrifuged at low speed to precipitate nuclei and chloroplasts. The intact mitochondria, being smaller in size than chloroplasts, remain in the supernatant. To get rid of nuclear DNA, one of two methods is usually used. In the first, the pellet containing nuclei and chloroplasts is treated with DNase. The latter has access to the nuclear DNA, via nuclear pores of the nuclear membrane, leading to its digestion, but has no access to chloroplast DNA because intact chloroplasts have a nonporous envelope. In the second method, the intact chloroplasts are banded in a sucrose— or a percoll—density—gradient, while the nuclei pellet. Banded chloroplasts are carefully removed. Intact chloroplasts, obtained by either a DNase method or a gradient method, are lysed and their proteins digested with a protease. Digested proteins are removed by organic (phenol/chloroform/isoamyl alcohol) extractions, and the nucleic acids (DNA and RNA) of the chloroplasts precipitated by ethanol.

Keywords

Intact Chloroplast Disodium EDTA Suspension Buffer Equivalent Rotor Swing Bucket Rotor 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Palmer, J.D., Downie, S.R., Nugent, J.M., Brandt, P., Unseld, M., Klein M., Brennicke, A., Schuster, W., and Börner, T. (1994) Chloroplast and mitochondrial DNAs of Arabidopsis thaliana: conventional genomes in an unconventional plant, in Arabidopsis Meyerowitz, E.M. and Somerville, C.R., eds.) pp. 37–62.Google Scholar
  2. 2.
    Bogorad, L., Gubbins, E.J., Krebbers, E., Larrinau, I.M., Mulligan, B.J., Muskuvitch, K.M.T., Orr, E.A., Rodermel, S.R., Schantz, R., Steinmetz, A.A., DeVos, G., and Ye, V.K. (1983) Cloning and physical mapping of maize plastid genes. Methods Enzymol. 97,524–555.CrossRefGoogle Scholar
  3. 3.
    Hermann, R,G., Bohnert, H.J., Kowallik, K.V., and Schmitt J.M. (1975) Size, confirmation and purity of chloroplast DNA from some higher plants. Biochim. Biophys. Acta 378, 305–31Google Scholar
  4. 4.
    Kolodner, R. and Yewari, K.K. (1975) The molecular size and confirmation of the chloroplast DNA from higher plants. Biochim. Biophys. Acta 402, 372–390.PubMedGoogle Scholar
  5. 5.
    Mourad, G. and Polacco, M. (1989) Mini-preparation of highly purified chloroplast DNA from maize. Plant Mol. Biol. Rep. 7, 78–84.CrossRefGoogle Scholar
  6. 6.
    Palmer, J.D. (1982) Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa. Nucleic Acids Res. 10,1593–1605.PubMedCrossRefGoogle Scholar
  7. 7.
    Fluhr, R. and Edelman, M. (1981) Physical mapping of Nicotiana tabacum chloroplast DNA. Mol. Gen. Genet. 181, 484–490.CrossRefGoogle Scholar
  8. 8.
    Tewari, K.K. and Wildman, S.G. (1966) Chloroplast DNA from tobacco leaves. Science 153, 1269–1271.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 1998

Authors and Affiliations

  • George S. Mourad
    • 1
  1. 1.Department of Molecular GeneticsJohn Innes CentreNorwichUK

Personalised recommendations