Real-Time Gap Ligase Chain Reaction

PURPOSE: We have developed a real-time semiquantitative gap ligase chain reaction for detecting p53 point mutations at low level in a background of excess of wild-type DNA. EXPERIMENTAL DESIGN: This method was validated by direct comparison to a previously validated but cumbersome phage plaque hybridization assay. Forty-one surgical margins and lymph nodes from 10 cases of head and neck squamous cell carcinoma and lung carcinoma were tested for p53 mutant clones. RESULTS: Both methods detected p53 mutants in margins from 8 of the 10 cases, whereas standard pathology detected cancer cells in only 3 cases. Positive margins included tissue samples with a tumor/normal DNA ratio of up to 1:1000. CONCLUSIONS: This novel molecular approach can be performed in <5 h facilitating intraoperative use for real-time surgical resection.