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 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.
Complete Metadata
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| description | 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. |
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| identifier | https://healthdata.gov/api/views/qgzn-g2df |
| issued | 2025-07-14 |
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| title | A modification of Representational Difference Analysis, with application to the cloning of a candidate in the Reelin signalling pathway |
