title: Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR.
abstract: Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies.
  
  
  CONCLUSIONS
  Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting.
authors.affiliation: IVIC, Tumor Cell Biology Laboratory, Apartado 21827, Caracas, 1020A, Venezuela.
authors.forename: Andrea
authors.initials: A
authors.lastname: Orue
authors.affiliation: IVIC, Tumor Cell Biology Laboratory, Apartado 21827, Caracas, 1020A, Venezuela.
authors.forename: Manuel
authors.initials: M
authors.lastname: Rieber
mesh_terms: D000483:Alleles; D011905:Genes, ras; D006801:Humans; D057230:Limit of Detection; D009154:Mutation; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*