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Nucleic Acids Res. 1985 Dec 20;13(24):8765-85.

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

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.

PMID:
3001650
PMCID:
PMC318950
DOI:
10.1093/nar/13.24.8765
[Indexed for MEDLINE]
Free PMC Article

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