Nucleic Acids Research, 1988, Vol. 16, No. 3 791-802
© 1988
Articles |
5'–3' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis
Max-Planck-Institut für Experimentelle Medizin Abteilung Chemie, Hermann-Rein-Strasse 3, D-3400 Göttingen, FRG
Received December 16, 1987. Accepted January 7, 1988.
The application of T7 and 
exonuclease to phosphorothioate-based oligonucletide-directed mutagenesis was investigated. Oligonucleotide primers designed to introduce single or double base mismatches, an insertion or a deletion (each of 16 bases) were annealed to M13 phage derivatives. Double stranded closed circular DNA (RF IV) containing phosphorothioate internucleotidic linkages in the (–)strand was prepared enzymatically from these templates. A nick was introduced into the (+)strand of the hetroduplex DNA. This nicked DNA (RF II) was subjected to treatment with T7 or exonuclease. Both of these enzymes were able to degrade almost all of the viral (+)strand when presented with DNA containing one or two base mismatches. Repolymerisation of the DNA after the gapping reaction, followed by transfection into E. coli cells gave mutational efficiencies of up to 95%. In the case of RF II DNA prepared with insertion or deletion primers these exonucleases could only partially degrade the viral (+)strand but were nevertheless highly efficient in such mutagenesis experiments.
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