Document Detail


K-ras point mutations in routinely processed tissues: non-radioactive screening by single strand conformational polymorphism analysis.
MedLine Citation:
PMID:  8157747     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
S H Korn; P T Moerkerk; A F de Goeij
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Journal of clinical pathology     Volume:  46     ISSN:  0021-9746     ISO Abbreviation:  J. Clin. Pathol.     Publication Date:  1993 Jul 
Date Detail:
Created Date:  1994-05-16     Completed Date:  1994-05-16     Revised Date:  2010-03-24    
Medline Journal Info:
Nlm Unique ID:  0376601     Medline TA:  J Clin Pathol     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  621-3     Citation Subset:  AIM; IM    
Affiliation:
Department of Pathology, University of Limburg Academic Hospital, Maastricht, The Netherlands.
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MeSH Terms
Descriptor/Qualifier:
Colonic Neoplasms / genetics*
DNA Mutational Analysis / methods
DNA, Neoplasm / analysis*
DNA, Single-Stranded / analysis*
Electrophoresis, Polyacrylamide Gel
Genes, ras / genetics*
Humans
Nucleic Acid Conformation
Paraffin Embedding
Point Mutation*
Polymerase Chain Reaction
Polymorphism, Genetic*
Chemical
Reg. No./Substance:
0/DNA, Neoplasm; 0/DNA, Single-Stranded
Comments/Corrections

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