Efficient site-directed mutagenesis by simultaneous use of two primers.
A rapid and efficient procedure for site specific mutagenesis is described. A double primed synthesis with a 17-mer mismatch primer and a "universal" 15-mer M13 sequencing primer was used to introduce a T to A transversion into an ompF signal peptide gene cloned in the M13mp8 vector. The two primers were annealed to the circular single stranded M13 template. After a short extension and ligation reaction, a double stranded restriction fragment containing the mismatch (ompF*/EcoR1-SalI) was cut out of the partly single stranded circular DNA and inserted into pBR322. 42% of the E.coli transformants harboured plasmid with the desired mutation, which could be detected by the appearance of a new restriction site (MboII) and by dot blot hybridization of plasmid DNA with the 32P-labeled 17-mer.
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D008565:Membrane Proteins / Q000235:genetics*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D013698:Templates, Genetic
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes