J. Aquiles Sanchez, Senior Research Scientist, Brandeis University
Numerous mutations in EGFR exons 18-21 determine sensitivity or resistance to tyrosine kinase inhibitors (TKIs) in many patients with Non-Small Cell Lung Carcinoma (NSCLC). Current technologies for detection of all such mutations utilize DNA sequencing or high resolution melting analysis, but interrogate only one exon at a time. We describe a convenient single closed-tube assay for simultaneous mutation scanning of all four EGFR exons. The assay utilizes two novel technologies invented in our laboratory: LATE-PCR, an advanced form of non-symmetric PCR and Lights-On/Lights-Off fluorescent probes. This multiplexed LATE-PCR assay simultaneously generates separate single-stranded amplicons for exons 18-21. The resulting products are 130-200 nucleotides long and each is probed at endpoint over a broad range of temperatures using a set of Lights-On/Lights-Off probes in a particular color. The signal from each bound Lights-On probe is quenched upon binding of an adjacent Lights-Off probe at a lower temperature. The resulting fluorescent signatures distinguish wild-type and silent mutations from TKI-related mutations with a sensitivity of 10% and a limit of detection down to single molecules. This single closed-tube assay runs in standard fluorescent thermocyclers, has proven reliable on genomic DNA from cultured cell lines, and is ready for validation with clinical samples. Funded by Smiths Detection Diagnostics, Inc grant to LJW.