Development of a facile fluorescent assay for the detection of 80 mutations within the p53 gene.
BACKGROUND
Alterations in the p53 tumor suppressor gene constitute one of the most frequent genetic events associated with the development of human cancers. Determination of an individual's p53 status may be of value in early diagnosis, prediction of response to treatment, and for the detection of minimal residual cancer. Recent studies have also revealed that specific mutations affecting the p53 gene are associated with a poor outcome. The majority of tumor biopsies that are sent for study in the laboratory contain neoplastic cells intermingled with stroma, such that the detection of alterations in the p53 gene requires a tumor enrichment technique and/or highly sensitive mutation detection technologies. Thus, it is desirable that a clinically useful assay for detecting point mutations in the p53 gene function in the presence of significant quantities of wild-type sequence and identify the critical sequence aberrations.


METHODS
We utilized molecular beacons in a real-time allele-specific PCR format to obtain reference data on samples of quantitatively known p53 mutation status. These data have been statistically analyzed and the results used to detect p53 mutations, indicating the presence of occult tumor.


RESULTS
We describe validation of a simple, rapid, sensitive, and quantitative ARMS assay for identifying the levels of 80 point mutations within the p53 gene that, when mutated, constitute at least 1% of the total p53 sequences.


CONCLUSIONS
The assay successfully identifies rare p53 gene mutations in clinical samples and overcomes many of the limitations of current technologies.
D001681:Biological Assay / Q000379:methods*; D015415:Biomarkers / Q000032:analysis; D002294:Carcinoma, Squamous Cell / Q000235:genetics; D005453:Fluorescence; D006801:Humans; D009062:Mouth Neoplasms / Q000235:genetics; D009154:Mutation; D016133:Polymerase Chain Reaction; D016159:Tumor Suppressor Protein p53 / Q000235:genetics*
D015415:Biomarkers; D016159:Tumor Suppressor Protein p53