MutS as a tool for mutation detection

• Autorzy: Stanislawska-Sachadyn A. , Sachadyn P.
• Języki publikacji: EN

Abstrakty

EN

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 µg 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 µg 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 β-galactosidase or GFP. Very low detection limits for β-galactosidase could theoretically enable mutation detection not only by the examination of PCR products, but even of genomic DNA.

Słowa kluczowe

EN

chimeric MutS protein; Thermus thermophilus; single nucleotide polymorphism; microbiology; mutation range; mutation detection; genetic disease; MutS protein; chimeric protein; fusion

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2005
• Tom: 52
• Numer: 3
• Opis fizyczny: p.575-583,fig.,ref.

Twórcy

autor

Stanislawska-Sachadyn A.

• Gdansk University of Technology, Gdansk, Poland

autor

Sachadyn P.

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