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Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences

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Summary

We have developed a selection procedure for mutants obtained by oligonucleotide directed mutagenesis based on asymmetrical A-methylation of GATC-sequences in the duplex DNA. The method involves the construction of gapped duplexes of circular single-stranded phage DNA. An oligonucleotide, complementary to part of the gap except for a single mismatch, is hybridized to the gapped duplex DNA and the remaining single stranded regions are filled-in enzymatically. When the template is undermethylated, the yield of mutants is almost, solely dependent on the priming efficiency of the oligonucleotide. The approach was used to introduce an AT→CG transversion in the nut L region of phage λ. Under optimal conditions, about 50–60% of the transformants were of the mutant genotype. Although situated adjacent to a known nut L mutation, the present mutation was phenotypically silent. The possibility of screening for mutants by means of a coupled, easily detectable marker was also investigated.

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Abbreviations

bp:

base pairs

RF:

replicative form

ssDNA:

single stranded DNA

Ap gene:

carbenicillin resistance gene

EtBr:

ethidium bromide

O.D.:

optical density

Kb:

kilobases

PL :

major leftward promoter of phage λ

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Authors and Affiliations

  1. Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat 35, B-9000, Ghent, Belgium

    Anne Marmenout, Erik Remaut & Walter Fiers

  2. Department of Organic Chemistry, State University of Leiden, P.O. Box 9502, 2300 RA, Leiden, The Netherlands

    Jacques van Boom

Authors
  1. Anne Marmenout
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  2. Erik Remaut
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  3. Jacques van Boom
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  4. Walter Fiers
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Communicated by W. Arber

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Marmenout, A., Remaut, E., van Boom, J. et al. Oligonucleotide directed mutagenesis: Selection of mutants by hemimethylation of GATC-sequences. Mol Gen Genet 195, 126–133 (1984). https://doi.org/10.1007/BF00332734

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  • DOI: https://doi.org/10.1007/BF00332734

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