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Nucleic Acids Research, 1988, Vol. 16, No. 14 6987-6999
© 1988

Articles

Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro

Hans-Joachim Fritz*, Joachim Hohlmaier+, Wilfried Kramer§, Andrea Ohmayer and Jürgen Wippler

Max-Planck-Institut für Biochemie Abteilung Zeilbiologie, Am Klopferspitz 18a, D-8033 Martinsried bei München, FRG

*To whom correspondence should be addressed

Received March 2, 1988. Revised June 14, 1988. Accepted June 14, 1988.

The gapped duplex DNA approach to oligonucleotide-directed construction of mutations (Kramer et al. 1984, Nucl. Acids Res. 12, 9441–9456) has been developed further. A procedure is described that makes in vitro DNA polymerase/DNA ligase reactions dispensable. Direct transfection of host bacteria with gdDNA molecules of recombinant phage M13 plus mutagenic oligonucleotide results in marker yields in excess of 50% (gap size 1640 nucleotides). An important feature incorporated into the mutagenic oligonucleotide is the presence of one or two intemucleotidic phosphorothioate linkages immediately adjacent to the 5'-terminus. Automated preparation and biochemical properties of such compounds are described as well as their performance in oligonucleotide-directed mutagenesis. A systematic study of the following parameters influencing marker yield is reported: Gap size, length of oligonucleotide, chemical nature of oligonucleotide termini and heatshock temperature during transformation.

+Dr. Müller-Lierheim AG, Behringstraße 6, D-8033 Planegg / München, FRG

§University of California, Department of Genetics, 345 Mulford Hall Berkeley, CA 94720, U.S.A


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