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. 1988 Jul 25;16(14B):6987–6999. doi: 10.1093/nar/16.14.6987

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

H J Fritz 1, J Hohlmaier 1, W Kramer 1, A Ohmayer 1, J Wippler 1
PMCID: PMC338346  PMID: 3405755

Abstract

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 internucleotidic 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.

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Selected References

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