Methodology article

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

Anelia A Kuvbachieva¹ and Andre M Goffinet²

¹  Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium

²  Developmental Genetics Unit, Univ. Louvain Med. School, Avenue E. Mounier, 73 Box 82, B1200 Brussels, Belgium

author email corresponding author email

BMC Molecular Biology 2002, 3:6doi:10.1186/1471-2199-3-6

Published:	24 April 2002

Abstract

Background

cDNA-RDA is one of the subtractive cloning techniques used to isolate differentially expressed genes between two complex cDNA populations. In the present study we present a modification of the protocol described by Hubank and Schatz.

Results

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.

Conclusion

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.