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Nucleic Acids Research, Vol 25, Issue 17 3552-3554, Copyright © 1997 by Oxford University Press

ARTICLES

Rapid determination of the complexity of cDNA bands extracted from DDRT- PCR polyacrylamide gels

NR Smith, A Li, M Aldersley, AS High, AF Markham and PA Robinson
Molecular Medicine Unit, St James's University Hospital, University of Leeds, Leeds LS9 7TF, UK. rmrnrs@stjames.leeds.ac.uk

A band extracted from a differential display polyacrylamide gel often represents a composite of heterogeneous products. We have developed a non- radioactive method to simply and rapidly analyse its complexity. A fluorescent restriction enzyme fingerprint of the composite mixture is generated. The number of individual bands observed in this fingerprint indicates the complexity of the re-amplified cDNA mixture. Restriction fingerprints of the inserts of cDNA subclones derived from the re- amplified cDNA mixture are compared to the composite fingerprint to select those representing the most intense bands in the composite. This dramatically reduces the number of clones required for further characterisation.
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