Abstract
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.
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References
Vogelstein, B. and Kinzler, K. W. (1993) The multistep nature of cancer. Trends Genet. 9, 138–141.
Lisitsyn, N., Lisitsyn, N., and Wigler, M. (1993) Cloning the differences between two complex genomes. Science 259, 946–951.
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.
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.
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.
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.
Hubank, M. and Schatz, D. G. (1994) Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucl. Acids Res. 22, 5640–5648.
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.
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.
Lisitsyn, N. and Wigler, M. (1995) Representational difference analysis in the detection of genetic lesions in cancer. Methods Enzymol. 254, 291–304.
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.
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.
Lewin, B. (1994) Genome size and genetic content, in Genes V. Oxford: Oxford University Press, pp. 657–676.
Strauss, D. and Ausubel, F. M. (1990) Genomic subtraction for cloning DNA corresponding to deletion mutations. Proc. Natl. Acad. Sci. USA 87, 1889–1893.
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.
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.
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.
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.
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.
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Hollestelle, A., Schutte, M. (2005). Representational Difference Analysis as a Tool in the Search for New Tumor Suppressor Genes. In: Su, G.H. (eds) Pancreatic Cancer. Methods in Molecular Medicine™, vol 103. Humana Press. https://doi.org/10.1385/1-59259-780-7:143
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DOI: https://doi.org/10.1385/1-59259-780-7:143
Publisher Name: Humana Press
Print ISBN: 978-1-58829-107-3
Online ISBN: 978-1-59259-780-2
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