Open AccessSemiquantitative Chemiluminescent Detection of UV-B-Induced Point Mutations in the p53 Tumor-Suppressor Gene
Author(s) -
Wilfried Siefken,
U. Hoppe,
Jörg Bergemann
Publication year - 1999
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/99263cr01
Subject(s) - hacat , point mutation , microbiology and biotechnology , biology , mutant , genomic dna , southern blot , gene , allele , dna , mutation , variants of pcr , blot , polymerase chain reaction , serial dilution , genetics , cell culture , medicine , alternative medicine , pathology
The technique of allele-specific PCR (AS-PCR) enables the detection of a small number of mutant alleles in a large number of wild-type (WT) alleles. We used the AS-PCR technique and Southern blotting, using a nonradioactive labeled probe to analyze the formation of point mutations in the tumor-suppressor gene p53 of primary keratinocytes after UV-B irradiation. These permanent mutations resulting from CC dimers occur at distinct “hot-spots”, one of which is affected in the human keratinocyte cell line HaCaT. This enabled us to establish the method with a defined positive control template, which also allowed semiquantitative determination of the mutation frequency. This, and the determination of the detection limit, was done with the use of serial dilutions of WT genomic DNA from primary keratinocytes with mutant genomic HaCaT DNA in the AS-PCR assay.