Two buffer PAGE system-based SSCP/HD analysis: a general protocol for rapid and sensitive mutation screening in cystic fibrosis and any other human genetic disease
| Autor: | Richard Kraemer, Sabina Liechti-Gallati, Dieter Neeser, Vreni Schneider |
|---|---|
| Rok vydání: | 1999 |
| Předmět: |
Cystic Fibrosis
Sequence analysis Population Buffers Biology medicine.disease_cause Sensitivity and Specificity Frameshift mutation Genetics medicine Humans Missense mutation education Polymorphism Single-Stranded Conformational Genetics (clinical) DNA Primers Mutation education.field_of_study Base Sequence Point mutation Genetic Diseases Inborn Single-strand conformation polymorphism Molecular biology Electrophoresis Polyacrylamide Gel Heteroduplex |
| Zdroj: | European Journal of Human Genetics. 7:590-598 |
| ISSN: | 1476-5438 1018-4813 |
| Popis: | The large size of many disease genes and the multiplicity of mutations complicate the design of an adequate assay for the identification of disease-causing variants. One of the most successful methods for mutation detection is the single strand conformation polymorphism (SSCP) technique. By varying temperature, gel composition, ionic strength and additives, we optimised the sensitivity of SSCP for all 27 exons of the CFTR gene. Using simultaneously SSCP and heteroduplex (HD) analysis, a total of 80 known CF mutations (28 missense, 22 frameshift, 17 nonsense, 13 splicesite) and 20 polymorphisms was analysed resulting in a detection rate of 97.5% including the 24 most common mutations worldwide. The ability of this technique to detect mutations independent of their nature, frequency, and population specificity was confirmed by the identification of five novel mutations (420del9, 1199delG, R560S, A613T, T1299I) in Swiss CF patients, as well as by the detection of 41 different mutations in 198 patients experimentally analysed. We present a three-stage screening strategy allowing analysis of seven exons within 5 hours and analysis of the entire coding region within 1 week, including sequence analysis of the variants. Additionally, our protocol represents a general model for point mutation analysis in other genetic disorders and has already been successfully established for OTC deficiency, collagene deficiency, X-linked myotubular myopathy (XLMTM), Duchenne and Becker muscular dystrophy (DMD, BMD), Wilson disease (WD), Neurofibromatosis I and II, Charcot-Marie-Tooth disease, hereditary neuropathy with liability to pressure palsies, and defects in mitochondrial DNA. No other protocol published so far presents standard SSCP/HD conditions for mutation screening in different disease genes. |
| Databáze: | OpenAIRE |
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