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Northern and Southern Blotting and the Polymerase Chain Reaction to Detect Gene Expression

  • Thelma Tennant
  • Marina Chekmareva
  • Carrie W. Rinker-Schaeffer
Part of the Methods in Molecular Medicine book series (MIMM, volume 57)

Abstract

For cancer cells to form a metastasis, cells from the primary tumor must overcome the local adhesive forces, migrate and invade the microcirculation, arrest at a secondary site, and then finally proliferate (1). As implied by its mult]step nature, cancer metastasis is a complex and dynamic process that is likely to be regulated by a series of genes at each step (2). A variety of approaches have been used to discern the molecular events that regulate this process. It is likely that the ability of a cancer cell to form clinically detectable metastases is influenced by a variety of factors, including alt]rations in the pattern of gene expression within the cancer cell. Such changes could be the result]of genetic or epigenetic modifications (3). Alt]ough there has been a growing emphasis on array-based techniques for high-throughput screening of gene expression patterns, there are several well established protocols that can be used to identify such molecular changes. This chapter describes two of these techniques: Northern and Southern blotting.

E. M. Southern first described a method for immobilizing size-fractionated DNA fragments on a nitrocellulose membrane in 1975. Since then, a number of different variations of this blotting method have been developed, as well as a variety of ways by which scientists can generate and hybridize probes to detect specifically the sequences thus immobilized. Southern blotting is now a general term for a number of different methods by which DNA is transferred from a gel to a membrane, and because nitrocellulose is relatively fragile, improved membranes have been developed that are more durable and that have been optimized for allowing binding of nucleic acids.

Keywords

Sodium Dodecyl Sulfate Wash Solution Capillary Transfer Hybridization Oven Plastic Wrap 
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.
    Killion, J. J. and Fidler, I. J. (1989) The biology of tumor metastasis. Semin. Oncol. 16, 106–115.PubMedGoogle Scholar
  2. 2.
    Yoshida, B. A., Chekmareva, M. A., Wharam, J. F., Kadkhodaian, M., Stadler, W. M., Boyer, A., et al. (1998) Prostate cancer metastasis-suppressor genes: acurrent prospective. In Vivo 12, 49–5PubMedGoogle Scholar
  3. 3.
    Baylin, S. B., Herman, J. G., Graff, J. R., Vertino, P. M., and Issa, J. P. (1998)Alt]rations in DNA methylation: a fundamental aspect of neoplasia. Adv. CancerRes. 72, 141–196.CrossRefGoogle Scholar
  4. 4.
    Southern, E. M. (1975) Detection of specific sequences among DNA fragmentsseparated by gel electrophoresis. J. Mol. Biol. 98, 503–517.CrossRefPubMedGoogle Scholar
  5. 5.
    Bird, A. P. (1986) CpG-rich-islands and the function of DNA methylation. Nature 321, 209–213.CrossRefPubMedGoogle Scholar
  6. 6.
    Counts, J. L. and Goodman, J. I. (1995) Altrations in DNA methylation may playa variety of roles in carcinogenesis. Cell 83, 13–15.CrossRefPubMedGoogle Scholar
  7. 7.
    Gonzalez-Zulueta, M. G., Bender, C. M., Yang, A. S., Nguyen, T., Beart, R. W., Van Tornout, J. M, and Jones, P. A. (1995) Methylation of the 5' CpG island ofthe p16/CDKN2 tumor suppressor gene in normal and transformed tissues correlateswith gene silencing. Cancer Res. 55, 4531–4535.PubMedGoogle Scholar
  8. 8.
    Herman, J. G., Merlo, A., Mao, L., Lapidus, R. G., Issa, J. P. J, Davdson, N. E., et al.(1995) Inactivation of the CDKN2/p16/MTS1 gene is frequently associated with aberrant DNA methylation in all common cancers. Cancer Res. 55, 4525–2530.PubMedGoogle Scholar
  9. 9.
    Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350–5354.CrossRefPubMedGoogle Scholar
  10. 10.
    Schroeder, M. and Mass, M. J. (1997) CpG methylation inactivates the transcriptional activity of the promoter of the human p53 tumor suppressor gene. Biochem. Biophy. Res. Commun. 235, 403–406.CrossRefGoogle Scholar
  11. 11.
    Pharmacia-Biotech mRNA Purification Kit Instructions. (1998) Revision 4.Google Scholar
  12. 12.
    Amersham Life Science Megaprime DNA Labeling Systems Instruction Manual. (1998) RPN 1604/5/6/7.Google Scholar
  13. 13.
    Bio-Rad Zeta-Probe Membranes Instruction Manual. (1998).2–4, 10-17, 25-26.Google Scholar
  14. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1998) Current Protocols in Molecular Biology, Vols. 1-3. John Wiley and Sons,.2.1–3.Google Scholar
  15. Bachman, K. E., Herman, J. G., Corn, P. G., Merlo, A., Costello, J. F., Cavanee, W. K., et al. (1999) Methylation-associated silencing of the tissue inhibitor of metalloproteinase-3 gene suggest a suppressor role in kidney, brain, and other human cancers. Cancer Res. 4, 798–802.Google Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Thelma Tennant
    • 1
  • Marina Chekmareva
    • 1
  • Carrie W. Rinker-Schaeffer
    • 1
  1. 1.University of ChicagoChicago

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