A fast, sensitive and accurate high resolution melting (HRM) technology-based assay to screen for common K-ras mutations.

摘要: Background: Increasing evidence points to a negative correlation between K-ras mutations and patient's response to, or survival benefit after, treatment with EGFR-inhibitors. Therefore, rapid reliable assays for mutational analysis of the gene are strongly needed. Methods: We designed high resolution melting (HRM) technology-based approach followed by direct sequencing deter- mine exon 1 (codons 12/13) tumour genotype. Results: Reconstruction experiments demonstrated an analytical sensitivity HRM assay following se- quencing 1.5-2.5% mutated DNA in background wild-type DNA. Assay reproducibility accuracy were 100%. Application onto genomic isolated from formalin-fixed paraffin-embedded specimens non-small cell lung cancer (n = 91) colorectal 7) patients revealed nucleotide substitutions at codons 12 13, including homozygous mutation, 33 (34%) 5 (5%) cases, respectively. Comparison conventional nested-PCR cycle-sequencing showed overall agreement genotype findings (kappa value 0.96), more detected sequencing. Conclusions: allows rapid, sensitive pre-screening routine diagnostic subsequent genotyp- ing mutations, even if present low abundance homozygosity, may considerably facilitate personalized therapy planning.