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


CONCLUSIONS
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.
Kuvbachieva
Anelia A
AA
Neurobiology Unit 2853, Univ. Namur Med. Sch., 61, rue de Bruxelles, Namur, Belgium. kuv1302@yahoo.co.uk
Goffinet
Andre M
AM