Affiliations: [a] Center for Translational Genetics, B. Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology and Rambam Health Care Campus, Haifa, Israel | [b] Institute of Pathology, Rambam Health Care Campus, Haifa, Israel | [c] B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel | [d] Legacy Heritage Clinical Research Institute at Rambam Health Care Campus, Haifa, Israel
Correspondence:
[*]
Corresponding author: Dov Hershkovitz, Institute of Pathology, Rambam Health Care Campus, PO Box 9602, Haifa 31096, Israel. Tel.: +972 485 425 30; Fax: +972 485 432 54; E-mail: [email protected].
Abstract: Introduction:KRAS mutations in colon carcinomas are associated with lack of response to anti-EGFR monoclonal antibody treatment. Therefore, patients must undergo genetic testing to be eligible for treatment. Several methods for KRAS mutation analysis exist, but many are not sensitive enough to detect a mutation in samples with low fraction of malignant cells. In the present study, we developed a KRAS mutations detection method that is both simple and sensitive. Methods:Using a locked nucleic acid (LNA) containing oligonucleotide, we developed a PCR clamping method that preferentially amplifies the mutated over wild type KRAS. We evaluated the sensitivity of this method using serial dilutions of plasmids containing wild-type and mutated KRAS fragments. Additionally, KRAS mutation status was evaluated on 60 archived tissue samples of colon carcinoma, and compared to direct sequencing and high resolution melting (HRM) methods. Results:The PCR clamping method could detect as little as 1% mutated DNA in the sample analyzed. Of the 29 KRAS mutations identified by the PCR clamping method, only 23 (79%) were identified by standard direct sequencing. The results of PCR clamping correlated with HRM results. Conclusions:LNA based PCR clamping method is a simple and highly sensitive method for the detection of KRAS mutations.
