Representational Difference Analysis as a Tool in the Search for New Tumor Suppressor Genes

  • Antoinette Hollestelle
  • Mieke Schutte
Part of the Methods in Molecular Medicine™ book series (MIMM, volume 103)


The recognition of a homozygous deletion of genetic material in a tumor genome has been instrumental in several tumor suppressor gene searches. The representational difference analysis (RDA) allows one to identify homozygous deletions even from among the high background of allelic losses that is typical for most cancers. RDA is a polymerase chain reaction (PCR)-based subtractive hybridization method. Two major obstacles to successful enrichment of target sequences from complex genomes were circumvented by RDA. Incomplete reassociation of complex DNA populations is overcome by using representative subpopulations of the tester and driver genomes. In addition, reiterated hybridization, selection, and amplification of the difference products introduces a kinetic component in the enrichment of target sequences. RDA thus enables the identification of homozygous deletions as small as 100 kilobases. Here, we provide a detailed protocol of the RDA procedure, including reflections on frequently encountered technical problems and on the particulars of its application in cancer.

Key Words

Allelic loss gene identification homozygous deletion kinetic enrichment protocol representational difference analysis subtractive hybridization tumor suppressor gene 


  1. 1.
    Vogelstein, B. and Kinzler, K. W. (1993) The multistep nature of cancer. Trends Genet. 9, 138–141.PubMedCrossRefGoogle Scholar
  2. 2.
    Lisitsyn, N., Lisitsyn, N., and Wigler, M. (1993) Cloning the differences between two complex genomes. Science 259, 946–951.PubMedCrossRefGoogle Scholar
  3. 3.
    Lisitsyn, N. A., Lisitsina, N. M., Dalbagni, G., et al. (1995) Comparative genomic analysis of tumors: Detection of DNA losses and amplification. Proc. Natl. Acad. Sci. USA 92, 151–155.PubMedCrossRefGoogle Scholar
  4. 4.
    Schutte, M., da Costa, L. T., Hahn, S. A., et al. (1995) Identification by representational difference analysis of a homozygous deletion in pancreatic carcinoma that lies within the BRCA2 region. Proc. Natl. Acad. Sci. USA 92, 5950–5954.PubMedCrossRefGoogle Scholar
  5. 5.
    Li, J., Yen, C., Liaw, D., et al. (1997) PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 275, 1943–1947.PubMedCrossRefGoogle Scholar
  6. 6.
    Zeschnigk, M., Horsthemke, B., and Lohmann, D. (1999) Detection of homozygous deletions in tumors by hybridization of representational difference analysis (RDA) products to chromosome-specific YAC clone arrays. Nucl. Acids Res. 27, e30.PubMedCrossRefGoogle Scholar
  7. 7.
    Hubank, M. and Schatz, D. G. (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucl. Acids Res. 22, 5640–5648.PubMedCrossRefGoogle Scholar
  8. 8.
    Welford, S. M., Gregg, J., Chen, E., et al. (1998) Detection of differentially expressed genes in primary tumor tissues using representational difference analysis coupled to microarray hybridization. Nucl. Acids Res. 26, 3059–3065.PubMedCrossRefGoogle Scholar
  9. 9.
    Toyota, M., Ho, C., Ahuja, N., et al. (1999) Identification of differentially methylated sequences in colorectal cancer by methylated CpG island amplification. Cancer Res. 59, 2307–2312.PubMedGoogle Scholar
  10. 10.
    Lisitsyn, N. and Wigler, M. (1995) Representational difference analysis in the detection of genetic lesions in cancer. Methods Enzymol. 254, 291–304.PubMedCrossRefGoogle Scholar
  11. 11.
    Lamar, E. E. and Palmer, E. (1984) Y-encoded, species-specific DNA in mice: Evidence that the Y chromosome exists in two polymorphic forms in inbred strains. Cell 37, 171–177.PubMedCrossRefGoogle Scholar
  12. 12.
    Kunkel, L. M., Monaco, A. P., Middlesworth, W., Ochs, H. D., and Latt, S. A. (1985) Specific cloning of DNA fragments absent from the DNA of a male patient with an X chromosome deletion. Proc. Natl. Acad. Sci. USA 82, 4778–4782.PubMedCrossRefGoogle Scholar
  13. 13.
    Lewin, B. (1994) Genome size and genetic content, in Genes V. Oxford: Oxford University Press, pp. 657–676.Google Scholar
  14. 14.
    Strauss, D. and Ausubel, F. M. (1990) Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA 87, 1889–1893.CrossRefGoogle Scholar
  15. 15.
    Wieland, I., Bolger, G., Asouline, G., and Wigler, M. (1990) A method for difference cloning: Gene amplification following subtractive hybridization. Proc. Natl. Acad. Sci. USA 87, 2720–2724.PubMedCrossRefGoogle Scholar
  16. 16.
    Kinzler, K. W. and Vogelstein, B. (1989) Whole genome PCR: Application to the identification of sequences bound by gene regulatory proteins. Nucl. Acids Res. 17, 3645–3653.PubMedCrossRefGoogle Scholar
  17. 17.
    Bishop, D. T., Williamson, J. A., and Skolnick, M. H. (1983) A model for restriction fragment length distributions. Am. J. Hum. Genet. 35, 795–815.PubMedGoogle Scholar
  18. 18.
    Schutte, M., Rozenblum, E., Moskaluk, C. A., et al. (1995) An integrated high-resolution physical map of the DPC/BRCA2 region at chromosome 13q12. Cancer Res. 55, 4570–4574.PubMedGoogle Scholar
  19. 19.
    Schutte, M., da Costa, L. T., Moskaluk, C. A., et al. (1995) Isolation of YAC insert sequences by representational difference analysis. Nucl. Acids Res. 23, 4127–4133.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Antoinette Hollestelle
    • 1
  • Mieke Schutte
    • 1
  1. 1.Department of Medical OncologyJosephine Nefkens Institute, Erasmus MCRotterdamThe Netherlands

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