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Updated: Jun 12, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
A novel mutant-enriched liquidchip technology for the qualitative detection of somatic mutations in KRAS gene from
Shiyang Wu1, Zeyao Zhu, Jiaying He
1SurExam Bio-Tech Co. Ltd., Guangzhou Technology Innovation Base, Science City, Guangzhou, PR China.
Background:
Somatic mutations in the KRAS gene have been reported to confer drug resistance to epidermal growth factor receptor tyrosine kinase inhibitors and some monoclonal antibodies. However, current DNA mutation detection technologies are primarily DNA sequencing-based and not high throughput, nor sensitive enough to meet clinical needs.
Methods:
A mutant-enriched PCR method was designed by introducing a unique restriction enzyme site to the PCR product. This allowed the wild-type KRAS sequence to be selectively removed by restriction enzyme digestion before application to the Luminex liquidchip system.
Results:
A total of 100 copies of mutant KRAS DNA fragment mixed with 1x10(5) copies of the wild-type KRAS DNA could be detected to achieve a sensitivity of 0.1%. This technology is currently used for clinical testing of KRAS somatic mutations for the purpose of pharmacogenomic evaluation. Serum samples from 109 patients with non-small cell lung cancer were tested and 34 mutations were detected (34/109). The formalin-fixed and paraffin-embedded samples from 60 patients with colorectal cancer were tested and 19 mutations were detected (19/60).
Conclusions:
A novel, qualitative, sensitive, reliable and high throughput liquidchip technology has been developed for detecting KRAS mutations using clinical serum and formalin-fixed and paraffin-embedded samples.
Insights
A new high-throughput method accurately detects KRAS mutations in cancer patients. This sensitive KRAS mutation test aids in pharmacogenomic evaluation for targeted therapies.
Area of Science:
- Molecular Biology
- Genetics
- Oncology
Background:
- Somatic mutations in the KRAS gene are linked to drug resistance against targeted cancer therapies.
- Existing DNA sequencing methods for KRAS mutation detection lack the necessary throughput and sensitivity for clinical applications.
Purpose of the Study:
- To develop a novel, high-throughput, and sensitive method for detecting KRAS mutations in clinical samples.
- To enable accurate pharmacogenomic evaluation for personalized cancer treatment.
Main Methods:
- A mutant-enriched PCR technique was employed, incorporating a unique restriction enzyme site.
- Wild-type KRAS sequences were selectively removed via restriction enzyme digestion.
- The processed samples were analyzed using the Luminex liquidchip system.
Main Results:
- The method achieved a sensitivity of 0.1%, detecting 100 copies of mutant KRAS DNA against 1x10(5) copies of wild-type DNA.
- Clinical testing on 109 non-small cell lung cancer serum samples identified 34 KRAS mutations.
- Testing on 60 colorectal cancer formalin-fixed, paraffin-embedded samples revealed 19 KRAS mutations.
Conclusions:
- A novel, sensitive, reliable, and high-throughput liquidchip technology for KRAS mutation detection has been successfully developed.
- This technology is suitable for clinical use with both serum and formalin-fixed, paraffin-embedded samples.
- The developed method supports pharmacogenomic evaluation for guiding cancer treatment decisions.
