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Nucleic Acids Research, 1983, Vol. 11, No. 15 5103-5112
© 1983

MOLECULAR BIOLOGY

Efficient site-directed mutagenesis by simultaneous use of two primers

Kjeld Norris, Fanny Norris, Lars Christiansen and Niels Fiil

Novo Research Institute, Novo Alle DK-2880 Bagsvaerd, Denmark

Received May 23, 1983. Revised May 23, 1983. Accepted July 11, 1983.

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.


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