The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA.
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.
D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics; D015246:Deoxyribonuclease EcoRI; D005090:Exodeoxyribonucleases / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods; D009154:Mutation; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D013873:Thionucleotides / Q000235:genetics*; D014170:Transformation, Genetic
D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D013873:Thionucleotides; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D004262:DNA Restriction Enzymes; D015246:Deoxyribonuclease EcoRI