The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA
- 1 January 1985
- journal article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 13 (24) , 8765-8785
- https://doi.org/10.1093/nar/13.24.8765
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.Keywords
This publication has 37 references indexed in Scilit:
- Convenient modification of the method for oligonucleotide‐directed in vitro mutagenesis of cloned DNAFEBS Letters, 1985
- Different base/base mismatches are corrected with different efficiencies by the methyl-directed DNA mismatch-repair system of E. coliCell, 1984
- Synthesis and characterization of an octanucleotide containing the EcoRI recognition sequence with a phosphorothioate group at the cleavage siteBiochemistry, 1984
- [32] Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectorsPublished by Elsevier ,1983
- [31] Directed mutagenesis with sodium bisulfitePublished by Elsevier ,1983
- [4] Exonuclease III: Use for DNA sequence analysis and in specific deletions of nucleotidesPublished by Elsevier ,1983
- [2] New M13 vectors for cloningPublished by Elsevier ,1983
- Site-specific mutagenesis by error-directed DNA synthesisNature, 1982
- Site-specific mutagenesis using oligodeoxyribonucleotides: isolation of a phenotypically silent φX174 mutant, with a specific nucleotide deletion, at very high efficiencyGene, 1980
- Site-directed mutagenesis in DNA: Generation of point mutations in cloned β globin complementary DNA at the positions corresponding to amino acids 121 to 123Journal of Molecular Biology, 1978