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Updated: Jun 8, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Detection of DNA mutations by fluorescence resonance energy transfer-based preferential homoduplex formation assay
Shiro Kitano1, Mashimo Nakayama, Akio Yamane
1Life Science Research Laboratory, Toppan Printing, Kisarazu, Chiba 292-0818, Japan. shiro.kitano@toppan.co.jp
Abstract:
Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET-PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET-PHFA detected KRAS mutations (1.25-50%) from all cell line DNA titration samples.
Insights
A new fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA) offers a rapid and cost-effective method for detecting cancer-driving KRAS mutations. This assay shows high reproducibility and sensitivity for companion diagnostics in molecularly targeted cancer therapy.
Area of Science:
- Oncology
- Molecular Biology
- Biotechnology
Background:
- Molecularly targeted cancer therapies are increasingly popular but require companion diagnostics to assess target molecule status.
- Accurate detection of target molecule status (expression, activity, mutation) is crucial for therapeutic efficacy.
- Existing methods for somatic mutation detection can be complex, costly, and time-consuming.
Purpose of the Study:
- To develop and evaluate a simple, cost-effective, and rapid somatic mutation detection method.
- To assess the performance of the developed assay for detecting KRAS mutations in codons 12 and 13.
- To compare the novel assay with a commercially available clinical diagnostic kit.
Main Methods:
- Development of the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET-PHFA).
- Utilized double-stranded labeled DNA and fluorescence measurement with thermal control.
- Evaluated FRET-PHFA for detecting multiallelic KRAS mutations in codons 12 and 13 using cell line DNA titration samples.
Main Results:
- FRET-PHFA demonstrated higher reproducibility, easier handling, and reduced contamination risk compared to conventional methods.
- The assay achieved a significantly shorter assay time (approximately 15 minutes) and lower cost.
- FRET-PHFA successfully detected KRAS mutations at concentrations ranging from 1.25% to 50% in all tested cell line DNA titration samples.
Conclusions:
- FRET-PHFA is a highly sensitive, reproducible, and cost-effective method for somatic mutation detection.
- The assay is suitable for companion diagnostics, particularly for identifying KRAS mutations prior to targeted cancer therapy.
- FRET-PHFA offers a valuable alternative to existing methods, enabling faster and more accessible molecular profiling for cancer treatment.
