Analysis of the amplification refractory mutation allele-specific polymerase chain reaction system for sensitive and specific detection of p53 mutations in DNA.

Abstract

The sensitivity of the amplification refractory mutation allele-specific polymerase chain reaction system (ARMAS-PCR) to detect known p53 mutations was determined using DNA extracted from two human tumour cell lines collected by cytobrush, as a model for its use in exfoliative cytology. Using DNA extracted from SW480 and CEM cell lines diluted with normal human fibroblasts, a nested ARMAS-PCR was more sensitive than a non-nested version and could detect one mutated cell amongst 100 000 normal cells. When compared with PCR-single stranded conformational polymorphism, nested ARMAS-PCR was 10 000 times more sensitive for detecting mutant p53 in extracted DNA. Primer design proved to be influential on the sensitivity and specificity of the assay; increased specificity was achieved by the use of deliberate mismatches upstream from the 3' end of mutation-specific primers. ARMAS-PCR was confirmed to be specific for the mutation that each primer was designed to detect. Nested ARMAS-PCR offered a rapid and sensitive method of analysis of cells with predetermined p53 mutations and has the potential to be applied to the study of the molecular progression of cancer, including diagnosis and detection of residual disease. It could also be extended to the in situ detection of aberrant cells.