Preliminary studies on DNA retardation by MutS applied to the detection of point mutations in clinical samples
| Autor: | Maria Sromek, Józef Kur, Aneta Balabas, Paweł Sachadyn, Zygmunt Paszko, Anna Kluska, Aneta Janiec-Jankowska, Anna Stanisławska-Sachadyn, Alicja Wiśniewska, Elzbieta Skasko |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
congenital
hereditary and neonatal diseases and abnormalities Health Toxicology and Mutagenesis Pcr cloning Genes BRCA1 Cell Cycle Proteins Multiple Endocrine Neoplasia Type 2b chemistry.chemical_compound Proto-Oncogene Proteins Genetics Point Mutation Mutation detection Nucleotide Molecular Biology Gene chemistry.chemical_classification Oncogene Proteins biology Point mutation Proto-Oncogene Proteins c-ret Nuclear Proteins Receptor Protein-Tyrosine Kinases DNA Thermus thermophilus biology.organism_classification Molecular biology DNA-Binding Proteins MutS Homolog 2 Protein chemistry Heteroduplex |
| Zdroj: | Mutation research. 570(1) |
| ISSN: | 0027-5107 |
| Popis: | MutS ability to bind DNA mismatches was applied to the detection of point mutations in PCR products. MutS recognized mismatches from single up to five nucleotides and retarded the electrophoretic migration of mismatched DNA. The electrophoretic detection of insertions/deletions above three nucleotides is also possible without MutS, thanks to the DNA mobility shift caused by the presence of large insertion/deletion loops in the heteroduplex DNA. Thus, the method enables the search for a broad range of mutations: from single up to several nucleotides. The mobility shift assays were carried out in polyacrylamide gels stained with SYBR-Gold. One assay required 50–200 ng of PCR product and 1–3 μg of Thermus thermophilus his 6 -MutS protein. The advantages of this approach are: the small amounts of DNA required for the examination, simple and fast staining, no demand for PCR product purification, no labelling and radioisotopes required. The method was tested in the detection of cancer predisposing mutations in RET , hMSH2 , hMLH1 , BRCA1 , BRCA2 and NBS1 genes. The approach appears to be promising in screening for unknown point mutations. |
| Databáze: | OpenAIRE |
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