A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway

In the post-hybridization mix, the 5'-ends of homoduplexes of interest (tester-tester) are filled-in with α-thio-deoxynucleotides. Unprotected duplexes, as well as the single-stranded DNA fragments, are degraded using ExoIII and Mung Bean Nuclease, prior to PCR subtraction, resulting in less complex difference products. We illustrate this modification by the cloning of a new gene which is differentially expressed in normal, reelin and Dab1 mutant mice and is a candidate member of the Reelin signalling pathway involved in brain development.We propose a modification of cDNA-RDA that may reduce the complexity of the post-hybridization mix and thus facilitate the amplification of differentially expressed products.Representational difference analysis of cDNA (cDNA-RDA) is a PCR-based subtractive hybridization technique for isolation and cloning of differentially expressed transcripts between two complex cDNA populations, without prior knowledge of their functional or biochemical characteristics [1,2]. The classical cDNA-RDA method, summarized in Fig. 1A, allows the selective amplification and cloning of transcripts that differ in abundance between two populations. Two complex cDNA populations are restricted with DpnII (restrictase that leaves 4-bp protruding 5' ends) and ligated to suitable linkers, the sequence of which is specific for the procedure. The amplicons, that represent the original RNA populations, are therefore called "representations" and provide the starting material. Next, the "tester" representation – from which isolation of specific messages is sought – is mixed with a large excess of the "driver" representation. After this subtractive hybridization, tester homoduplexes are enriched from the mixture by PCR (pre-PCR). Single strands are degraded using Mung Bean Nuclease (MBN), and a second PCR (final PCR) generates a complex population of amplicons that is named difference product. The procedure is usually repeated at least twice, increasing the ratio of