Objective: The aim of this study is to demonstrate possibility of detection of Ki-ras gene mutations using PCR clamped with a synthetic nucleotide analogue, locked nucleic acid (LNA). Setting: Institute of Clinical Biochemistry and Diagnostics, Faculty of Medicine Hradec Králové, Charles University Prague and University Hospital Hradec Králové; Department of Surgery, Faculty of Medicine Hradec Králové, Charles University Prague and University Hospital Hradec Králové; Department of General Surgery, Regional Hospital Pardubice; Department of Clinical Biochemistry, Regional Hospital Pardubice. Material and Methods: We examined seventy-three colorectal carcinomas (CRC) and plasma samples of twenty CRC patients for the presence of Ki-ras gene mutations in codon 12. PCR reactions were performed in Roche LightCycler System with hybridization probes and clamping LNA molecules. Results: Selective amplification of mutant gene sequences revealed Ki-ras gene changes in 22 cases (30%) of CRC. DNA sequencing confirmed GGT-GAT transition in 13 patients, GTT transversion in 8 patients, and AGT transition in one case. In our small experimental group of plasma DNA samples, however, we did not find any mutation in the Ki-ras gene. Most examined carcinomas were classified T2N0M0-T3N0M0, one carcinoma was T3N1M0. The detection limit of LNA-clamped PCR was about 2ng. Conclusion: We consider LNA-clamped real-time PCR to be a fast alternative method for specific analysis of the Ki-ras gene in colorectal carcinomas. The reliability determined in CRC tissues was comparable with the enriched PCR/RFLP and nucleotide sequencing.

        Key words: Ki-ras gene, point mutations, LNA, DNA, PCR, colorectal carcinoma, plasma.