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representational difference analysis (RDA)

A method for isolation and amplification of DNA sequences that differ between two populations (a subtractive technique); e.g. changes associated with antigenic variation in micro-organisms, activation of the immune system, chromosomal translocation. The complexity of one population, the tester DNA, is lowered by cleavage with an infrequent cutting endonuclease, ligated to adaptor oligonucleotides to allow PCR amplification; the other population, the driver DNA, is similarly treated, but after amplification the ligated adaptors are removed to prevent amplification. These amplicons do not include the entire parent population, but serve as representations. tester and an excess of driver DNA are mixed, denatured and annealed. By using kinetic enrichment conditions, the tester strands common to the two populations will be annealed to driver strands; only sequences unique to the tester population will form duplexes. The ends of the duplexes are filled in with a polymerase and PCR amplified; only fragments found uniquely in the tester population will amplify exponentially, as hybrid duplexes undergo only linear amplification and therefore will be a small fraction of the amplified fragments. Lisitsyn, N.A. (1995) Trends Genet. 11, 303-307

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