A rapid and efficient one-tube PCR-based mutagenesis technique using Pfu DNA polymerase

Nucleic Acids Res. 1994 Jul 11;22(13):2587-91. doi: 10.1093/nar/22.13.2587.


A rapid method for efficiently generating site-directed mutations on a clean sequence background is described. This modification of the megaprimer PCR mutagenesis approach can be performed in one tube in less than 4.5 hours, and does not require purification of intermediate products. High fidelity of DNA sequence replication is obtained by employing Pfu DNA polymerase and limiting the total number of amplification cycles to 30. The mutagenesis efficiency of the procedure is high enough to allow rapid, direct identification of mutants by restriction digest or sequencing techniques.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • DNA-Directed DNA Polymerase / metabolism*
  • Mutagenesis*
  • Polymerase Chain Reaction / methods*


  • Pfu DNA polymerase
  • DNA-Directed DNA Polymerase