[Detection of KRAS mutations in tumor cells using biochips].

ABSTRACT

Somatic mutations in the KRAS gene are important markers of some types of tumors, for example, pancreatic cancer, and may be useful in early diagnostics. A biochip has been developed which allows determining most frequent mutations in 12, 13 and 61 codons of the KRAS gene. To increase the sensitivity of the method and to make possible the analysis of minor fractions of tumor cells in clinical samples the method of blocking a wild type sequence PCR amplification by LNA-oligonucleotides has been used. The product of LNA-clamp PCR was further hybridized with oligonucleotide probes, immobilized on biochip. Biochip was tested with 42 clinical DNA samples from patients with pancreatic cancer, mostly ductal adenocarcinomas. As reference methods, the RFLP analysis and sequencing were used. The developed approach allows detecting somatic mutations in the KRAS gene if the portion of tumor cells with mutation is at least 1% of whole cell population.