Nucleic Acids Research, 1984, Vol. 12, No. 24 9441-9456
© 1984
MOLECULAR BIOLOGY |
The gapped duplex DNA approach to oligonucleotide-directed mutation construction
Institut für Genetik, Universität zu Köln Weyertal 121, D-5000 Köln 41 3Max-Planck-Institut für Molekulare Genetik Ihnestrasse 63, D-1000 Berlin 33, FRG
*To whom correspondence should be addressed
Received November 12, 1984. Accepted November 22, 1984.
A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (–)strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (–)strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (–)strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.
1Present address: Max-Planck-Institut für Biochemie, Abteilung Zellbiologie, Am Klopferspitz 2, D-8033 Martinsried, FRG
2Present address: A.N.Belozersky Laboratory of Moscow State University, Moscow, W-234, 119899, USSR
4Present address: Biozentrun der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
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