MutS as a tool for mutation detection

Acta Biochim Pol. 2005;52(3):575-83. Epub 2005 Aug 4.


MutS, a DNA mismatch-binding protein, seems to be a promising tool for mutation detection. We present three MutS based approaches to the detection of point mutations: DNA retardation, protection of mismatched DNA against exonuclease digestion, and chimeric MutS proteins. DNA retardation in polyacrylamide gels stained with SYBR-Gold allows mutation detection using 1-3 microg of Thermus thermophilus his6-MutS protein and 50-200 ng of a PCR product. The method enables the search for a broad range of mutations: from single up to several nucleotide, as mutations over three nucleotides could be detected in electrophoresis without MutS, due to the mobility shift caused by large insertion/deletion loops in heteroduplex DNA. The binding of DNA mismatches by MutS protects the complexed DNA against exonuclease digestion. The direct addition of the fluorescent dye, SYBR-Gold, allows mutation detection in a single-tube assay. The limited efficiency of T4 DNA polymerase as an exonuclease hampers the application of the method in practice. The assay required 300-400 ng of PCR products in the range of 200-700 bp and 1-3 microg of MutS. MutS binding to mismatched DNA immobilised on a solid phase can be observed thanks to the activity of a reporter domain linked to MutS. We obtained chimeric bifunctional proteins consisting of T. thermophilus MutS and reporter domains, like beta-galactosidase or GFP. Very low detection limits for beta-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.

Publication types

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

MeSH terms

  • Adenosine Triphosphatases / metabolism
  • Base Pair Mismatch / genetics*
  • DNA Mutational Analysis / methods*
  • Fluorescent Dyes
  • Genes, Reporter
  • Genetic Techniques
  • Genomics / methods
  • MutS DNA Mismatch-Binding Protein / metabolism*
  • MutS Homolog 2 Protein / metabolism*
  • Point Mutation*
  • Polymerase Chain Reaction
  • Thermus thermophilus / metabolism
  • Transplantation Chimera / genetics
  • beta-Galactosidase / metabolism


  • Fluorescent Dyes
  • beta-Galactosidase
  • Adenosine Triphosphatases
  • MutS DNA Mismatch-Binding Protein
  • MutS Homolog 2 Protein