Homogeneous amplification and mutation scanning of the p53 gene using fluorescent melting curves. Clin Chem 2002;48:1321–8

occur in tumor suppressor genes such as p53 and are sporadically located. We describe a homogeneous method for amplification and mutation scanning, and apply the method to the p53 gene. Methods: Using a series of overlapping fluorescein-labeled oligonucleotides complementary to a wild-type p53 sequence, we detected somatic mutations in colorec-tal cancers by aberrant probe:target melting tempera-tures (Tm). The probes were designed so that fluores-cence decreased on target annealing as a result of deoxyguanosine quenching. Probes were walked along the sequence to be scanned, using two to three probes per cuvette and placing overlapping probes in separate reactions. After amplification, the reaction was cooled to anneal probes and then slowly heated (0.1 °C/s) whil