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Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Optimized Multiplex Detection of 7 KRAS Mutations by Taqman Allele-Specific qPCR
1IVIC, Tumor Cell Biology Laboratory, Apartado 21827, Caracas, 1020A, Venezuela.
Unlabelled:
Establishing the KRAS mutational status of tumor samples is essential to manage patients with colorectal or lung cancer, since these mutations preclude treatment with monoclonal anti-epidermal growth factor receptor (EGFR) antibodies. We report an inexpensive, rapid multiplex allele-specific qPCR method detecting the 7 most clinically relevant KRAS somatic mutations with concomitant amplification of non-mutated KRAS in tumor cells and tissues from CRC patients. Positive samples evidenced in the multiplex assay were further subjected to individual allele-specific analysis, to define the specific mutation. Reference human cancer DNA harbouring either G12A, G12C, G12D, G12R, G12S, G12V and G13D confirmed assay specificity with ≤1% sensitivity of mutant alleles. KRAS multiplex mutation analysis usefulness was also demonstrated with formalin-fixed paraffin embedded (FFPE) from CRC biopsies.
Conclusion:
Co-amplification of non-mutated DNA avoided false negatives from degraded samples. Moreover, this cost effective assay is compatible with mutation detection by DNA sequencing in FFPE tissues, but with a greater sensitivity when mutant DNA concentrations are limiting.
Insights
This study presents a fast, affordable qPCR method to detect KRAS mutations in colorectal cancer (CRC) patients. The assay accurately identifies key mutations, aiding treatment decisions for anti-EGFR therapies.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- KRAS mutational status is critical for managing colorectal and lung cancer patients.
- Specific KRAS mutations limit the efficacy of anti-EGFR antibody treatments.
Purpose of the Study:
- To develop a cost-effective, rapid multiplex qPCR assay for detecting common KRAS mutations.
- To improve diagnostic accuracy for KRAS mutations in cancer patients.
Main Methods:
- A multiplex allele-specific qPCR method was designed to detect 7 clinically relevant KRAS mutations.
- The assay includes concomitant amplification of non-mutated KRAS to prevent false negatives.
- Positive samples underwent individual allele-specific analysis for precise mutation identification.
Main Results:
- The assay demonstrated high specificity, confirming the detection of G12A, G12C, G12D, G12R, G12S, G12V, and G13D mutations.
- Sensitivity for mutant alleles was as low as 1%.
- The method proved effective with formalin-fixed paraffin-embedded (FFPE) colorectal cancer biopsies.
Conclusions:
- Co-amplification of non-mutated DNA mitigates false negatives in degraded samples.
- The cost-effective assay is suitable for FFPE tissues and complements DNA sequencing.
- Offers enhanced sensitivity for detecting limited amounts of mutant DNA.
