K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis
| Autor: | A.F.P.M. de Goeij, S. H. Korn, P. T. M. Moerkerk |
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| Rok vydání: | 1993 |
| Předmět: |
DNA Mutational Analysis
DNA Single-Stranded Biology medicine.disease_cause Polymerase Chain Reaction Pathology and Forensic Medicine law.invention chemistry.chemical_compound law medicine Humans Point Mutation Gene Polymerase chain reaction COLD-PCR Mutation Paraffin Embedding Polymorphism Genetic Oligonucleotide Point mutation Single-strand conformation polymorphism DNA Neoplasm General Medicine Molecular biology Genes ras chemistry Colonic Neoplasms Nucleic Acid Conformation Electrophoresis Polyacrylamide Gel DNA Research Article |
| Zdroj: | Journal of Clinical Pathology. 46:621-623 |
| ISSN: | 0021-9746 |
| DOI: | 10.1136/jcp.46.7.621 |
| Popis: | AIMS--To develop a non-radioactive method to screen routinely fixed, paraffin wax embedded specimens for the occurrence of point mutations; to evaluate the single strand conformational polymorphism (SSCP) analysis technique for the detection of K-ras point mutations as a result of electrophoretic mobility shifts. METHODS--DNA was extracted from archival specimens of colon cancer and from established colon cancer cell lines with known point mutations. A K-ras gene fragment containing codons 12 and 13 of exon 1 was amplified with the polymerase chain reaction (PCR). Denatured DNA fragments were run on 10% polyacrylamide gels under non-denaturing conditions. After electrophoresis DNA was blotted and the single stranded DNA was detected using a digoxigenin labelled ras probe. The nature of the detected point mutations was identified and confirmed by sequencing and hybridisation with oligonucleotides using 32P labelling. RESULTS--Wild type and aberrant alleles were detected caused by mobility shifts after electrophoresis of the PCR products. Commonly occurring mutations in the K-ras gene--in the first two positions of codon 12--could easily be detected in DNA from archival paraffin wax embedded colon cancer tissue. In all the colon tumour samples studied wild type gene alleles were also found, presumably derived from normal cells in the specimen. CONCLUSIONS--The SSCP method permits rapid non-radioactive screening of adenomas or carcinomas for the occurrence of point mutations in the K-ras gene. But if a mutation is detected by an electrophoretic mobility shift, its identification requires confirmation by sequencing or oligonucleotide hybridisation. |
| Databáze: | OpenAIRE |
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