Advertisement

Isopycnic Centrifugation of DNA

Extraction and Fractionation of Melon Satellite DNA by Buoyant Density Centrifugation in Cesium Chloride Gradients
  • Donald Grierson
Protocol
Part of the Springer Protocols Handbooks book series (SPH)

Abstract

In order to study the organization and properties of DNA, it is necessary to extract it from cells and purify it from the proteins with which it is usually associated. Cells of plants and animals contain from 1 to 100 pg of DNA per nucleus. This does not sound like a lot until one realizes that is represents 100–10,000 times as much DNA as in an Escherichia coli chromosome. When faced with such vast complexity, it is obviously an advantage if the DNA can be separated into a number of more homogeneous fractions, thus simplifying genome analysis. One method of doing this is to exploit the density differences between various gene sequences by centrifuging the DNA in solutions of cesium salts, either alone or in the presence of various heavy metal ions, DNA-binding dyes, or antibiotics that modify density. When solutions of CsCl are centrifuged at sufficient speed, a concentration gradient is formed so that the solution at the bottom of the centrifuge tube is denser than that at the top. Under appropriate conditions, i.e., when the density range in the gradient corresponds to that of DNA, different gene sequences occupy different positions corresponding to their own density.

Keywords

Buoyant Density Cesium Chloride Cesium Salt Glass Centrifuge Tube Ultracentrifuge Tube 
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.
    Birnstiel, M., Speirs, J., Purdom, I., Jones, K., and Loening, U. E., (1968) Properties and composition of the isolated ribosomal DNA satellite of Xenopus laevis. Nature 219, 454–463.PubMedCrossRefGoogle Scholar
  2. 2.
    Flamm, W. G. (1972) Highly repetitive sequences of DNA in chromosomes. Int. Rev. Cytol. 32, 2–51.Google Scholar
  3. 3.
    Ingle, J., Pearson, G. G., and Sinclair, J. (1973) Species distribution and properties of nuclear satellite DNA in higher plants. Nature New Biol. 242, 193–197.PubMedCrossRefGoogle Scholar
  4. 4.
    Wells, R. and Ingle, J. (1970) The constancy of the buoyant density of chloroplast and mitochondrial deoxyribonucleic acid in a range of higher plants. Plant Physiol. 46, 178–179.PubMedCrossRefGoogle Scholar
  5. 5.
    Grierson, D. (1977) The Nucleus and the Organization and Transcription of Nuclear DNA, in The Molecular Biology of Plant Cells. (H. Smith, ed.) Blackwell, Oxford.Google Scholar
  6. 6.
    Sinclair, J., Wells, R., Deumling, B., and Ingle, J. (1975) The complexity of satellite deoxyribonucleic acid in a higher plant. Biochem. J. 149, 31–38.PubMedGoogle Scholar
  7. 7.
    Bendich, A. J. and Taylor, W. C. (1977) Sequence arrange ment in satellite DNA from the musk melon. Plant Physiol. 59, 604–609.PubMedCrossRefGoogle Scholar
  8. 8.
    Kadouri, A., Atsmon, D., and Edelman, M. (1975) Satellite-rich DNA in cucumber: Hormonal enhancement of synthesis and subcellular identification. Proc. Natl. Acad. Sci. USA 72, 2260–2264.PubMedCrossRefGoogle Scholar

Copyright information

© The Humana Press Inc. 1986

Authors and Affiliations

  • Donald Grierson
    • 1
  1. 1.Department of Physiology and Environmental SciencesUniversity of NottinghamLoughboroughUK

Personalised recommendations