Representational difference analysis of cDNA and genome comparisons

Abstract

Representational difference analysis (RDA) is a subtractive hybridization technique that combines polymerase chain reaction (PCR) amplification with kinetic enrichment to isolate DNA sequences present in one population, but not in another. For comparisons between bacterial genomes (genomic RDA), populations of chromosomal DNA restriction fragments (called “representations”) are used as the starting material. Defined oligonucleotide adapters are then ligated on to the 5'-ends of the DNA molecules, comprising one population (known as the “tester”) but not the other (the “driver”). As with RDA, complementary DNA (cDNA) RDA can be divided into a number of phases: the generation of representations (the PCR amplicons, representing the RNAs isolated from given bacterial populations), the PCR-coupled subtractive hybridization of the different representations, and the cloning and screening of the resultant products that represent the differences between the two populations that were compared. Detailed protocols for both RDA and cDNA RDA are described in the chapter.