Semiquantitative chemiluminescent detection of UV-B-induced point mutations in the p53 tumor-suppressor gene.
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.
Siefken
W
W
Beiersdorf AG, Hamburg, Germany.
Hoppe
U
U
Bergemann
J
J
D000483:Alleles; D002460:Cell Line; D004247:DNA / Q000032:analysis / Q000235:genetics; D016147:Genes, Tumor Suppressor / Q000235:genetics*; D006801:Humans; D008163:Luminescent Measurements; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000528:radiation effects; D014466:Ultraviolet Rays
D016159:Tumor Suppressor Protein p53; D004247:DNA