A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway.
(2002) BMC Molecular Biology — Vol. 3, p. 6 (2002)
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- 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.
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Kuvbachieva, A. A., & Goffinet, A. (2002). A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway. BMC Molecular Biology, 3, 6. https://doi.org/10.1186/1471-2199-3-6 (Original work published 2002)