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

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 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. 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.

journal_name

BMC Mol Biol

journal_title

BMC molecular biology

authors

Kuvbachieva AA,Goffinet AM

doi

10.1186/1471-2199-3-6

keywords:

subject

Has Abstract

pub_date

2002-04-24 00:00:00

pages

6

issn

1471-2199

journal_volume

3

pub_type

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