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Anal Biochem. 2009 Aug 15;391(2):166-8. doi: 10.1016/j.ab.2009.05.027. Epub 2009 May 21.

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

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  • 1UF Canc√©rologie Biologique et Bioth√©rapie, CHU Grenoble, 38 043 La Tronche, France.


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 approximately 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.

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