- Tytuł:
- MutS as a tool for mutation detection
- Autorzy:
-
Stanisławska-Sachadyn, Anna
Sachadyn, Paweł - Powiązania:
- https://bibliotekanauki.pl/articles/1041359.pdf
- Data publikacji:
- 2005
- Wydawca:
- Polskie Towarzystwo Biochemiczne
- Tematy:
-
fusion
SNP
chimeric protein
MutS
mutation detection - Opis:
- 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.
- Źródło:
-
Acta Biochimica Polonica; 2005, 52, 3; 575-583
0001-527X - Pojawia się w:
- Acta Biochimica Polonica
- Dostawca treści:
- Biblioteka Nauki
Artykuł