Homogeneous Amplification and Mutation Scanning of the p53 Gene Using Fluorescent Melting Curves

Abstract

In malignancy, gene mutations frequently 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. Using a series of overlapping fluorescein-labeled oligonucleotides complementary to a wild-type p53 sequence, we detected somatic mutations in colorectal cancers by aberrant probe:target melting temperatures (T(m)). The probes were designed so that fluorescence 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 degrees C/s) while fluorescence was continuously monitored. Somatic mutations in tumor tissue were detected by changes from a characteristic wild-type melting curve profile using leukocyte DNA. A complete scanning of the DNA binding domain (exons 5-8) of the p53 gene was completed in a single run ( approximately 30 min) starting from genomic leukocyte DNA. To show proof-of-principle, p53 exons 6-8 from 63 colon cancers were probe-scanned and showed 100% agreement with direct sequencing for detecting alterations from wild-type DNA. p53 mutation scanning by single-labeled hybridization probes is a homogeneous, rapid, and sensitive method with application in both research and clinical diagnostics.