Detection of Point Mutations in Kirsten ras 2 Gene Using Locked Nucleic Acids Clamped PCR
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a Department of clinical biochemistry and diagnostics, Faculty of Medicine and Faculty Hospital Hradec Králové, b Department of internal medicine, Faculty of Medicine and Faculty Hospital Hradec Králové, c Department of surgery, Regional Hospital Pardubice, d Department of surgery, Faculty of Medicine and Faculty Hospital Hradec Králové, e Department of medical biology and genetics, Faculty of Medicine and Faculty Hospital Hradec Králové, f TIB MOLBIOL, Berlin, Germany
The aim of the study was to examine diagnostic possibilities of LNA (locked nucleic acids) -clamped PCR for detection of somatic mutations in the K-ras gene in 133 samples of human malignant colorectal tumors. Selective real-time PCR amplification of mutant alleles of the K-ras gene (codon 12 and 13) revealed the presence of mutations in 45 samples of the tumors. The detection limit of the used method for K-ras mutant alleles was 0.03 ng. LNA-clamped PCR suppressing amplification of the wild-type alleles of the K-ras gene provides a very sensitive and specific detection of mutations present in DNA samples of colorectal carcinomas.