The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA

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ID: 302540
1985
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Abstract
M13 RF IV DNA may be prepared in vitro to contain phosphorothioate-modified internucleotidic 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 M13mp2 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.
Reference Key
openalex_W2042869569 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors W. John Taylor, Johann Ott, Fritz Eckstein
Journal Nucleic Acids Research
Year 1985
DOI
10.1093/nar/13.24.8765
URL
https://doi.org/10.1093/nar/13.24.8765
Keywords Keywords not found

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