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MutS as a tool for mutation detection

100%

Stanisławska-Sachadyn A., Sachadyn P.

Acta Biochimica Polonica

2005

vol. 52

issue 3

575-583

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.

Thermal profile with alternately raised and lowered annealing temperature improves the PCR amplification using highly degenerate primers

81%

Sachadyn P., Sobiewska G., Kur J.

Acta Biochimica Polonica

1998

vol. 45

issue 3

691-694

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2 Acta Biochimica Polonica

1 2005

1 1998

2 Sachadyn P.

1 Kur J.

1 Sobiewska G.

1 Stanisławska-Sachadyn A.

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MutS as a tool for mutation detection

Thermal profile with alternately raised and lowered annealing temperature improves the PCR amplification using highly degenerate primers