title: LNA-based PCR clamping enrichment assay for the identification of KRAS mutations.
abstract: BACKGROUND
  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.
authors: 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.
authors: Edna
authors: E
authors: Efrati
authors: Hela
authors: H
authors: Elkin
authors: Yehudit
authors: Y
authors: Peerless
authors: Edmond
authors: E
authors: Sabo
authors: Ofer
authors: O
authors: Ben-Izhak
authors: Dov
authors: D
authors: Hershkovitz
mesh_terms: D004252:DNA Mutational Analysis / Q000401:mortality*; D006801:Humans; D009841:Oligonucleotides / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D012680:Sensitivity and Specificity; D018631:ras Proteins / Q000235:genetics*
chemical_list: C117307:KRAS protein, human; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; C477371:locked nucleic acid; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins