A modified two primer approach to oligonucleotide-directed in vitro mutagenesis

P. F G Sims, S. J. Minter, R. Stancombe, M. E. Gent, J. Andrews, R. B. Waring, P. Towner, R. W. Davies

    Research output: Contribution to journalArticlepeer-review

    Abstract

    Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E. coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L. casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila. We have used a modification of the method described by Norris et al. [1]. A mutagenic primer and an M13 universal sequencing primer are annealed simulaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors. We have analysed the effect on the frequency of mutation of:o1)the temperature used for strand extension;2)the class of base change attempted;3)the host mismatch repair system. A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies. © 1985 Masson, Paris.
    Original languageEnglish
    Pages (from-to)841-847
    Number of pages6
    JournalBiochimie
    Volume67
    Issue number7-8
    DOIs
    Publication statusPublished - Jul 1985

    Keywords

    • catabolite activator protein
    • mismatch repair
    • oligonucleotide-directed mutagenesis
    • RNA splicing

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