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lüll MutS as a tool for mutation detection Stanislawska-Sachadyn A; Sachadyn PActa Biochim Pol 2005[]; 52 (3): 575-83MutS, 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.|*Point Mutation[MESH]|Adenosine Triphosphatases/metabolism[MESH]|Base Pair Mismatch/*genetics[MESH]|DNA Mutational Analysis/*methods[MESH]|Fluorescent Dyes[MESH]|Genes, Reporter[MESH]|Genetic Techniques[MESH]|Genomics/methods[MESH]|MutS DNA Mismatch-Binding Protein/*metabolism[MESH]|MutS Homolog 2 Protein/*metabolism[MESH]|Polymerase Chain Reaction[MESH]|Thermus thermophilus/metabolism[MESH]|Transplantation Chimera/genetics[MESH]|beta-Galactosidase/metabolism[MESH] |