SDM-Assist software to design site-directed mutagenesis primers introducing "silent" restriction sites

BMC Bioinformatics. 2013 Mar 22;14:105. doi: 10.1186/1471-2105-14-105.

Abstract

Background: Over the past decades site-directed mutagenesis (SDM) has become an indispensable tool for biological structure-function studies. In principle, SDM uses modified primer pairs in a PCR reaction to introduce a mutation in a cDNA insert. DpnI digestion of the reaction mixture is used to eliminate template copies before amplification in E. coli; however, this process is inefficient resulting in un-mutated clones which can only be distinguished from mutant clones by sequencing.

Results: We have developed a program - 'SDM-Assist' which creates SDM primers adding a specific identifier: through additional silent mutations a restriction site is included or a previous one removed which allows for highly efficient identification of 'mutated clones' by a simple restriction digest.

Conclusions: The direct identification of SDM clones will save time and money for researchers. SDM-Assist also scores the primers based on factors such as Tm, GC content and secondary structure allowing for simplified selection of optimal primer pairs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Arabidopsis Proteins / genetics
  • DNA Primers / chemistry*
  • DNA Restriction Enzymes
  • Escherichia coli / genetics
  • Mutagenesis, Site-Directed / methods*
  • Polymerase Chain Reaction / methods
  • Potassium Channels / genetics
  • Software*

Substances

  • Arabidopsis Proteins
  • DNA Primers
  • Potassium Channels
  • AKT1 protein, Arabidopsis
  • DNA Restriction Enzymes