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Published on: September 25, 2018
LNA-based PCR clamping enrichment assay for the identification of KRAS mutations
Edna Efrati1, Hela Elkin, Yehudit Peerless
1Center for Translational Genetics, B. Rappaport Institute for Research in the Medical Sciences, Technion-Israel Institute of Technology and Rambam Health Care Campus, Haifa, Israel.
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.
Insights
A new locked nucleic acid (LNA) based PCR clamping method offers a simple and highly sensitive approach for detecting KRAS mutations in colon carcinomas, improving diagnostic accuracy.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutations in colon cancer predict poor response to anti-EGFR therapies, necessitating genetic testing for treatment eligibility.
- Existing KRAS mutation detection methods often lack the sensitivity required for samples with low tumor cell fractions.
- Accurate KRAS mutation analysis is crucial for personalized treatment strategies in colorectal cancer.
Purpose of the Study:
- To develop a simple and highly sensitive method for detecting KRAS mutations in colon carcinoma.
- To improve the detection rate of KRAS mutations, especially in samples with low malignant cell content.
Main Methods:
- Development of a PCR clamping technique utilizing locked nucleic acid (LNA) oligonucleotides.
- Preferential amplification of mutated KRAS sequences over wild-type sequences.
- Evaluation of sensitivity using serial dilutions and comparison with direct sequencing and high-resolution melting (HRM) on 60 archived colon carcinoma samples.
Main Results:
- The LNA-based PCR clamping method demonstrated high sensitivity, detecting mutations present at 1% in DNA samples.
- This method identified 29 KRAS mutations, with 23 (79%) also detected by standard direct sequencing.
- PCR clamping results showed good correlation with high-resolution melting (HRM) analysis.
Conclusions:
- Locked nucleic acid (LNA) based PCR clamping is a simple and highly sensitive method for KRAS mutation detection.
- This technique offers improved diagnostic capabilities for KRAS mutations in colorectal cancer specimens.
- The method holds potential for enhancing patient selection for targeted therapies.
