Rapid determination of the complexity of cDNA bands extracted from DDRT-PCR polyacrylamide gels.
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.
Smith
N R
NR
Molecular Medicine Unit, St James's University Hospital, University of Leeds, Leeds LS9 7TF, UK. rmrnrs@stjames.leeds.ac.uk
Li
A
A
Aldersley
M
M
High
A S
AS
Markham
A F
AF
Robinson
P A
PA
D001483:Base Sequence; D016172:DNA Fingerprinting; D004262:DNA Restriction Enzymes; D018076:DNA, Complementary / Q000032:analysis* / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase
D018076:DNA, Complementary; D005452:Fluoresceins; D005456:Fluorescent Dyes; C024098:6-carboxyfluorescein; D012194:RNA-Directed DNA Polymerase; D004262:DNA Restriction Enzymes