title: Pyrosequencing method to detect KRAS mutation in formalin-fixed and paraffin-embedded tumor tissues.
abstract: 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.
authors.affiliation: UF Cancérologie Biologique et Biothérapie, CHU Grenoble, 38 043 La Tronche, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Dufort
authors.forename: Marie-Jeanne
authors.initials: MJ
authors.lastname: Richard
authors.forename: Florence
authors.initials: F
authors.lastname: de Fraipont
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D018631:ras Proteins / Q000235:genetics*
chemical_list: D005404:Fixatives; D005557:Formaldehyde; D066246:ErbB Receptors; D018631:ras Proteins