Semiquantitative chemiluminescent detection of UV-B-induced pointmutations in the p53 tumor-suppressor gene

摘要

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 ASPCR 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.