Representational Difference Analysis

Abstract

Representational difference analysis (RDA) is a highly sensitive technique for the identification of differences between complex deoxyribonucleic acid samples. RDA is based on polymerase chain reaction amplification in combination with multiple rounds of subtractive hybridization and can be used for the identification of unknown pathogens, genetic lesions in cancer and polymorphic markers linked to a trait without the use of a preexisting map, as well as for the identification of differentially expressed genes.

Keywords: RDA; representational difference analysis; amplicon; tester; driver; subtraction; subtractive hybridization; kinetic enrichment; differential expression; PCR; polymerase chain reaction; DNA; RNA; cDNA

Figure 1.

Schematic overview of representational difference analysis of complementary DNAs (cDNA‐RDA) for the identification of differentially expressed genes. ds: double‐stranded.

Figure 2.

Effect of suppression of previously identified products in representational difference analysis (RDA). Ethidium bromide staining of an agarose gel loaded with driver (D) and tester (T) DNA amplicons and the difference products obtained after one and two rounds of subtractive hybridization respectively. The dramatic effect of RDA is clear: the difference product 2 consists mainly of two major products (indicated by triangles), derived from genes highly expressed in the cells from which the tester was derived only. A disadvantage of this very high level of purification of these two gene fragments is the fact that many other differentially expressed genes are underrepresented or absent in the difference product 2. However, cDNA‐RDA offers the unique possibility to suppress the reappearance of previously identified products by adding those to the driver in a new RDA experiment. Difference products obtained after 1 (S1), 2 (S2) and 3 (S3) rounds of subtractive hybridization are shown; the two previously identified products are completely absent, and many additional differentially expressed genes can be identified this way (for details, see Groot and van Oost ).

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Further Reading

Everts RE, Versteeg SA, Renier C, et al. (2000) Isolation of DNA markers informative in purebred dog families by genomic representational difference analysis (gRDA). Mammalian Genome: Official Journal of the International Mammalian Genome Society 11: 741–747.

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Groot, Peter C, and van Oost, Bernard A(Sep 2005) Representational Difference Analysis. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0005374]