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Numerous mutations in exons 18-21 of the epidermal growth factor receptor (EGFR) gene determine the response of many patients with Non-Small Cell Lung Carcinoma (NSCLC) to anti-EGFR Tyrosine Kinase Inhibitors (TKIs). This paper describes a single closed-tube assay for simultaneous mutational scanning of EGFR exons 18-21 using Linear-After-The-Exponential (LATE)-PCR and Lights-On/Lights-Off probes. The assay readily detects mutations which cause sensitivity or resistance to TKIs and can distinguish these clinically important genetic changes from silent mutations which have no impact on protein function. The assay identifies as little as 5% mutant sequences in mixtures of normal DNA and mutant DNA prepared from cancer cell lines. Proof-of-principle experiments demonstrate mutation identification in formalin-fixed, paraffin-embedded NSCLC biopsies. The LATE-PCR EGFR assay described here represents a new type of highly informative, singletube diagnostic test for mutational scanning of multiple gene coding regions and/or multiple gene targets for personalized cancer therapies.
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Last date updated on June, 2014