A positive selection vector for cloning of long polymerase chain reaction fragments based on a lethal mutant of the crp gene of Escherichia coli

We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is succ...

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Bibliographic Details
Published in:Analytical biochemistry 1998-03, Vol.257 (2), p.203-209
Main Authors: Schlieper, D, von Wilcken-Bergmann, B, Schmidt, M, Sobek, H, Müller-Hill, B
Format: Article
Language:English
Subjects:
Cloning, Molecular - methods
Cyclic AMP Receptor Protein - genetics
DNA, Bacterial - biosynthesis
DNA, Bacterial - genetics
Escherichia coli - genetics
Escherichia coli - metabolism
Genes, Bacterial
Genes, Lethal
Genetic Vectors - genetics
Humans
Mutation
Polymerase Chain Reaction - methods
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Summary:We have constructed a cloning vector with a tight positive selection for recombinant clones in Escherichia coli. The positive selection pressure results from a lethal mutation within the E. coli gene coding for the catabolite gene activator protein CAP, which is disrupted whenever a fragment is successfully inserted. Here, we show that this "suicide" vector, pCAPs, is suitable for cloning of PCR products as long as 9.3 kb into several unique restriction sites which are scattered throughout the lethal gene.
ISSN:0003-2697
DOI:10.1006/abio.1997.2558