AAscan, PCRdesign and MutantChecker: a suite of programs for primer design and sequence analysis for high-throughput scanning mutagenesis

PLoS One. 2013 Oct 30;8(10):e78878. doi: 10.1371/journal.pone.0078878. eCollection 2013.


Scanning mutagenesis is a powerful protein engineering technique used to study protein structure-function relationship, map binding sites and design more stable proteins or proteins with altered properties. One of the time-consuming tasks encountered in application of this technique is the design of primers for site-directed mutagenesis. Here we present an open-source multi-platform software AAscan developed to design primers for this task according to a set of empirical rules such as melting temperature, overall length, length of overlap regions, and presence of GC clamps at the 3' end, for any desired substitution. We also describe additional software tools which are used to analyse a large number of sequencing results for the presence of desired mutations, as well as related software to design primers for ligation independent cloning. We have used AAscan software to design primers to make over 700 mutants, with a success rate of over 80%. We hope that the open-source nature of our software and ready availability of freeware tools used for its development will facilitate its adaptation and further development. The software is distributed under GPLv3 licence and is available at http://www.psi.ch/lbr/aascan.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arrestin / genetics
  • DNA Primers / genetics*
  • GTP-Binding Protein alpha Subunits, Gi-Go / genetics
  • Mutagenesis*
  • Polymerase Chain Reaction
  • Protein Engineering / methods*
  • Software*
  • User-Computer Interface


  • Arrestin
  • DNA Primers
  • GTP-Binding Protein alpha Subunits, Gi-Go

Grant support

This work was supported by the Swiss National Foundation grants 135754, 141898, 132815 and NCCR Structural Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.