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Nucleic Acids Research, 1989, Vol. 17, No. 12 4441-4454
© 1989

MOLECULAR BIOLOGY

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers

Patrick Stanssens*,, Chris Opsomer, Yvonne M. McKeown+,, Wilfried Kramer1,§, Marc Zabeau{pi}, and Hans-Joachim Fritz1,§,*

Plant Genetic Systems N.V. J. Plateaustraat 22, B-9000 Gent, Belgium 1Max-Planck-Institut fur Biochemie Abteilung Zellbiologie, Am Klopferspitz 18a, D-8033 Martinsried, FRG

*To whom correspondence should be addressed

Received April 18, 1989. Accepted May 25, 1989.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer el al. 1984, Nucl. Acids Res. 12, 9441–9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.

+Present addresses: Maclachlan & Donaldson, Patent Agents, 47 Merrion Square, Dublin 2, Ireland

§Present addresses: Institit für MOleckulare Genetik, Georg-August-Universität Göttingen, Grisetachstrase 8, D-3400 Gottingen

{pi}Present addresses: Helixm, Onafhankelijkheidslaan 38, B-9000, Gent, Belgium


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