Folia Microbiologica

, Volume 44, Issue 1, pp 11–14 | Cite as

PCR method for generating multiple mutations at adjacent sites

  • J. Adamec
  • F. Kalousek


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 delections 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 traditionalin vitro mutagenesis methods, one can generate large numbers of mutated plasmids in hours.


Multiple Mutation Adjacent Site Mutagenic Primer Unique Restriction Site External Primer 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


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Copyright information

© Institute of Microbiology, Academy of Sciences of the Czech Republic 1999

Authors and Affiliations

  1. 1.Institute of MicrobiologyAcademy of Sciences of the Czech RepublicPrague 4Czech Republic
  2. 2.Department of GeneticsYale School of MedicineNew HavenUSA

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