Nucleic Acids Research, 1985, Vol. 13, No. 24 8765-8785
© 1985
Articles |
The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA
Max-Planck-Institut für experimentelle Medizin, Abteilung Chemie Hermann-Rein-Strasse 3, D-3400 Göttingen, FRG
Received October 7, 1985. Revised November 26, 1985. Accepted November 26, 1985.
M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotldic linkages in the (–)strand only. Certain restriction enzymes react with this modified DNA to hydrolyze the (+)strand exclusively when a phosphorothioate linkage occurs at the normal cleavage point in the (–)strand. The reaction of Pvu I with M3mp2 RF IV DNA containing dCMPS residues in the (–)strand is of this type, and is exploited to allow subsequent digestion with exonuclease III of a portion of the (+)strand opposite different mutagenic mismatched oligonucleotide primers. Two methods are described by which this approach has been used to produce mutations in M13mp2 phage DNA with high efficiency as a result of simple and rapid in vitro manipulations. Plaques containing mutant phage in a genetically-pure form are obtained at a frequency of 40 – 66 %, allowing their characterisation directly by sequence analysis without prior screening and plaque purification. The wide applicability of this approach is discussed.
1Present address: Laboratory of Bioorgsnic Chemistry and Biochemistry, The Rockefeller University, 1234) York Avenue, New York, NY 10021-6399, USA
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