MutS as a tool for mutation detection.
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.
Stanisławska-Sachadyn
Anna
A
Department of Microbiology, Gdańsk University of Technology, Gdańsk, Poland.
Sachadyn
Paweł
P
D000251:Adenosine Triphosphatases / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D005821:Genetic Techniques; D023281:Genomics / Q000379:methods; D051719:MutS DNA Mismatch-Binding Protein / Q000378:metabolism*; D051718:MutS Homolog 2 Protein / Q000378:metabolism*; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016963:Thermus thermophilus / Q000378:metabolism; D018183:Transplantation Chimera / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D005456:Fluorescent Dyes; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases; D051719:MutS DNA Mismatch-Binding Protein; D051718:MutS Homolog 2 Protein