19] Representational difference analysis in detection of genetic lesions in cancer

Abstract

This chapter presents a global approach to the analysis of the sequence differences between the genomes of the cancer and normal cells that uses a method called representational difference analysis (RDA). In principle, RDA can be used to derive probes for genomic losses, rearrangements, amplifications, point mutations, and pathogenic organisms found within the cancer cell. RDA belongs to the general class of DNA subtractive methodologies. These methodologies all have in common that one DNA population (the driver) is hybridized in excess against a second population (the tester) to remove common hybridizing sequences, thereby enriching sequences unique to the tester. RDA is a powerful, versatile, but complex procedure. To perform it properly requires not only diligence at the bench but also a clear headed understanding of how to use it. This chapter addresses both of these aspects. The chapter also discusses the application of RDA and provides detailed protocols. Pure subtractive methodologies have limited usefulness in the analysis of complex genomes. This is because the enrichment required to purify target sequences is very high and because the sequence complexity of DNA from higher organisms is too great for single copy sequences to hybridize sufficiently to completion. RDA combines subtractive enrichment with two further elements: kinetic enrichment and representation. The RDA procedure consists of the preparation of amplicons followed by multiple cycles of hybridization/amplification. Because RDA is a complex procedure, the inclusion of controls is controlled. Care and thought must be given to the source of DNA for analysis, and this is also discussed.