Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues

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KRAS mutation status has been reported to be a predictive marker of tumor response to epidermal growth factor receptor (EGFR) inhibitors. We have designed a pyrosequencing assay based on nested polymerase chain reaction (PCR) to characterize KRAS mutation status using formalin-fixed and paraffin-embedded tumor tissues. Mutant and wild-type KRAS cell lines were used to determine the specificity and sensitivity (detection limit ∼5% mutant alleles) of the method. The results obtained for tumor samples were 95% comparable to those obtained by dideoxy sequencing. Analysis of KRAS mutation using nested PCR and pyrosequencing is a simple, robust, fast, and sensitive method that can be used with formalin-fixed and paraffin-embedded tissues.