PCR method for generating multiple mutations at adjacent sites

J. Adamec, F. Kalousek

Research output: Contribution to journalArticlepeer-review


Procedures to introduce point mutations, restriction sites and insert or delete DNA fragments are very important tools to study protein function. We describe here two-step PCR-based method for generating single or multiple mutations, insertions and deletions in a small region of the sequence. In the first step, a unique restriction site is introduced near the part of DNA sequence to be changed, without changing the amino acid sequence. For this step, one of the methods already described can be used. In the second step, mutations are introduced using mutagenic primers containing the unique restriction site from the first step at the 5′ end, paired with a universal primer crossing another unique restriction site present originally in the sequence. The method is very simple, economic and rapid. In comparison with the traditional in vitro mutagenesis methods, one can generate large numbers of mutated plasmids in hours.

Original languageEnglish (US)
Pages (from-to)11-14
Number of pages4
JournalFolia Microbiologica
Issue number1
StatePublished - 1999
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology


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