Biological Procedures Online

, Volume 9, Issue 1, pp 18–26 | Cite as

Rapid sitrapid site-directed domain scanning mutagenesis of enteropathogenic Escherichia coli espD

Open Access


We developed a rapid mutagenesis method based on a modification of the QuikChange® system (Stratagene) to systemically replace endogenous gene sequences with a unique similar size sequence tag. The modifications are as follows: 1: the length of the anchoring homologous sequences of both mutagenesis primers were increased to 16 – 22 bp to achieve melting temperatures greater than 80°C. 2: the final concentrations of both primers were increased to 5–10 ng/µl and the final concentration of template to 1–2 ng/µl. 3: the annealing temperature was adjusted when necessary from 52°C to 58°C. We generated 25 sequential mutants in the cloned espD gene (1.2 kb), which encodes an essential component of the type III secretion translocon required for the pathogenesis of enteropathogenic E. coli (EPEC) infection. Each mutation consisted of the replacement of 15 codons (45 bp) with 8 codons representing a 24 bp sequence containing three unique restriction endonuclease sites (KpnI/MfeI/SpeI) starting from the second codon. The insertion of the restriction endonuclease sites provides a convenient method for further insertions of purification and/or epitope tags into permissive domains. This method is rapid, site-directed and allows for the systematic creation of mutants evenly distributed throughout the entire gene of interest.

Indexing terms

Mutagenesis, Site-Directed Polymerase Chain Reaction Plasmids Sequence Deletion 



base pair


enteropathogenic Escherichia coli


percentage guanine plus cytosine DNA content




polyacrylamide gel electrophoresis


melting temperature


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Copyright information

© Springer 2007

Authors and Affiliations

  • Qiwen Deng
    • 1
  • Wensheng Luo
    • 1
  • Michael S. Donnenberg
    • 1
  1. 1.Division of Infectious Diseases, Department of MedicineUniversity of Maryland School of MedicineBaltimoreUSA

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