Two-stage PCR protocol allowing introduction of multiple mutations, deletions and insertions using QuikChange Site-Directed Mutagenesis

Biotechniques. 1999 Apr;26(4):680-2. doi: 10.2144/99264st03.


We developed a two-stage procedure, based on the QuikChange Site-Directed Mutagenesis Protocol, that significantly expands its application to a variety of gene modification experiments. A pre-PCR, single-primer extension stage before the standard protocol allows the efficient introduction of not only point mutation but also multiple mutations and deletions and insertions to a sequence of interest.

MeSH terms

  • Ampicillin Resistance / genetics
  • Base Sequence
  • Biotechnology
  • DNA Primers / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional
  • Mutagenesis, Site-Directed*
  • Plasmids / genetics
  • Point Mutation
  • Polymerase Chain Reaction / methods*
  • Sequence Deletion


  • DNA Primers