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Phytoplasma pp 381-393 | Cite as

Cesium Chloride-Bisbenzimide Gradients for Separation of Phytoplasma and Plant DNA

  • Lucy T. T. Tran-NguyenEmail author
  • Bernd Schneider
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 938)

Abstract

CsCl–bisbezimide gradient centrifugation is a method to separate phytoplasma from host plant DNA. Bisbenzimide forms a complex with A + T-rich DNA thereby lowering its relative density. During centrifugation the A + T-rich phytoplasma DNA is spatially separated from the less A + T-rich host plant DNA. The difference in buoyant density between phytoplasma DNA and plant DNA varies according to the host–pathogen combination. The phytoplasma DNA forms a distinct band above the host plant DNA and can be collected. Depending on the phytoplasma titer and the scale of extraction, highly purified DNA is obtained in sufficient quantities for the construction of a genomic library, a sequencing project or hybridization studies.

Key words

Bisbenzimide Buoyant density gradient centrifugation Cesium chloride DNA extraction Hoechst 33258 

Notes

Acknowledgements

The authors would like to thank Erich Seemüller (Julius Kühn-Institute, Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and Viticulture) for critically reading the chapter.

References

  1. 1.
    Schildkraut CL, Marmur J, Doty P (1962) Determination of the base composition of deoxyribonucleic acid from its buoyant density in CsCl. J Mol Biol 4:430–443PubMedCrossRefGoogle Scholar
  2. 2.
    Bontemps H, Houssier C, Fredericq E (1975) Physio-chemical study of the complexes of “33258 Hoechst” with DNA and nucleohistone. Nucleic Acids Res 2:971–984PubMedCrossRefGoogle Scholar
  3. 3.
    Manuelidis L (1977) A simplified method for preparation of mouse satellite DNA. Anal Biochem 78:561–568PubMedCrossRefGoogle Scholar
  4. 4.
    Burckhardt G et al (1985) Two binding modes of netropsin are involved in the complex formation with poly(dA-dT).poly(dA-dT) and other alternating DNA duplex polymers. J Biomol Struct Dyn 2:721–736PubMedCrossRefGoogle Scholar
  5. 5.
    Kopka ML et al (1985) Binding of an antitumor drug to DNA, Netropsin and C-G-C-G-A-A-T-T-BrC-G-C-G. J Mol Biol 183:553–563PubMedCrossRefGoogle Scholar
  6. 6.
    Kollar A, Seemüller E (1989) Base composition of the DNA of mycoplasma-like organisms associated with various plant diseases. J Phytopathol 127:177–186CrossRefGoogle Scholar
  7. 7.
    Kube M et al (2008) The linear chromosome of the plant-pathogenic mycoplasma ‘Candidatus Phytoplasma mali’. BMC Genomics 9:306PubMedCrossRefGoogle Scholar
  8. 8.
    Tran-Nguyen LTT et al (2008) Comparative genome analysis of ‘Candidatus Phytoplasma australiense’ (subgroup tuf Australia I; rp-A) and ‘Ca. Phytoplasma asteris’ strains OY-M and AY-WB. J Bacteriol 190:3979–3991PubMedCrossRefGoogle Scholar
  9. 9.
    Berg M, Seemüller E (1999) Chromosomal organization and nucleotide sequence of the genes coding for the elongation factors G and Tu of the apple proliferation phytoplasma. Gene 226:103–109PubMedCrossRefGoogle Scholar
  10. 10.
    Capesius I et al (1975) An A + T-rich satellite DNA in a monocotyledonous plant, Cymbidium. Biochim Biophys Acta 395:67–73PubMedCrossRefGoogle Scholar
  11. 11.
    Sacristán MD, Dobrigkeit I (1973) Variable content of a (GC) rich satellite DNA in tumorous and normal cultures of Crepis capillaris. Z Naturforschung C 28:564–567Google Scholar
  12. 12.
    Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  13. 13.
    Ahrens U, Seemüller E (1992) Detection of DNA of plant pathogenic mycoplasma-like organisms by a polymerase chain reaction that amplifies a sequence of the 16S rRNA gene. Phytopathology 82:828–832CrossRefGoogle Scholar
  14. 14.
    Porebski S, Bailey LG, Baum BR (1997) Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components. Plant Mol Biol Rep 15:8–15CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  1. 1.Department of ResourcesPlant IndustriesDarwinAustralia
  2. 2.Julius Kühn-Institute (JKI), Federal Research Centre for Cultivated Plants, Institute for Plant Protection in Fruit Crops and ViticultureDossenheimGermany

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