Detection of KRAS mutations in tumor cells using biochips

Molecular Biology (Impact Factor: 0.72). 10/2011; 45(5):797-803. DOI: 10.1134/S0026893311040042


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 deter-mining most frequent mutations in 12, 13, and 61 codons of the KRAS gene. To increase the sensitivity of the method and to enable the analysis of minor fractions of tumor cells in clinical
samples, the method of blocking 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 the biochip. The biochip was tested with
42 clinical DNA samples from patients with pancreatic cancer, mostly duct adenocarcinomas. As reference methods, 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 the whole cell population.

Keywordssomatic mutations–tumor–
KRAS gene–biochips

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    ABSTRACT: Somatic mutations of KRAS, PIK3CA, and BRAF cause insensitivity of colorectal tumors to therapy with anti-EGFR monoclonal antibodies, necessitating a genetic testing prior to therapy. A biological microchip was developed and validated to allow detection of 19 somatic mutations in KRAS, PIK3CA, and BRAF genes. The method combines LNA-clamp PCR and allele-specific hybridization on a microchip and detects mutant DNA in 100 times wild-type background (1%). A total of 66 DNA samples isolated from colorectal tumors were tested with the biochip. Possible associations between the genetic status of the tumor and the patient's characteristics (age, sex, tumor localization, stage, and TMN) were assessed statistically. KRAS mutations were more common in females (P = 0.02) and in patients with distant metastasis (P = 0.04). Other associations between the presence of mutations and patient characteristics were not observed. The method proved highly sensitive and can be used in oncology to select patients who sensitive to therapy with anti-EGFR monoclonal antibodies.
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