Detection of KRAS mutation by Hybri-probe

Abstract

The epidermal growth factor receptor (EGFR) is activated through self-phosphorylation by extracellular stimuli and transmits the signal to a downward signal pathway. Mutation of the EGFR is suggested to be closely related to the promotion of oncogenesis and metastasis. Anti-EGFR antibody therapy is effective for unresectable, advanced, or recurrent colon cancer, and is covered by medical insurance. However, the effectiveness of the anti-EGFR antibody is not confirmed in a patient who has a mutation of KRAS that resides in the downward signal pathway of the EGFR. Thus, KRAS mutation analyses may be important to predict the effectiveness of the anti-EGFR antibody treatment for a patient with EGFR mutation. Mutations at codon 12 of KRAS from the body cavity fluid of such patient was amplified by PCR and subjected to cloning using a plasmid. The complete discrimination of some mutants was difficult by our hybridization probe (Hybri-Probe) method using a wild-type probe (GGT). Therefore, we employed a mutant-specific probe (GAT) in addition to the wild-type probe, and the complete discrimination of five types of mutant and wild-type alleles was successful. The detection sensitivity (or minimal copy number for the detection) of our Hybri-Probe method was 1.0 × 10³ copy/μL, and the selection sensitivity was GGT:GAT (80:20), or at least 20% of the mutant [GAT] is necessary to discriminate from wild-type alleles [GGT]. Our method is simple, rapid and cheap, and expected to contribute to clinical settings.