Related Experiment Video
Updated: Mar 26, 2026
Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
Published on: September 25, 2018
Detection of KRAS mutations using double-stranded toehold-exchange probes
Zhenhua Wu1, Tianle Ma2, Jean-Luc Coll3
1State Key Laboratory of Transducer Technology, Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences, Shanghai 200050, China; University of Chinese Academy of Sciences, Beijing 100039, China.
Abstract:
Detection of KRAS mutations in cancer tissues is immensely valuable for the identification of personalized genotype-based therapy. Here, we employed a double-stranded toehold-exchange probe, which is labeled with fluorescent molecules (FAM) and quenchers (Dabcyl), to detect KRAS mutations in cancer tissues. This probe was able to differentiate the intended mutation in a sample containing as little as 5% mutant alleles in a background of wild-type DNA. This probe also performed robustly at a wide range of conditions, for examples, from 4 °C to 37 °C, from 200 mM Na(+) to 1M Na(+), and from 200 mM K(+) to 500 mM K(+). Furthermore, we validated the practicality of this probe in a clinical setting using 8 pairs of cancer tissue samples and their NT (corresponding adjacent nontumorous tissue) samples. All the results generated from the probe detection agreed with those from direct sequencing. Combining features of extreme high specificity and robustness, this probe is a valuable tool for reliable diagnosis of cancer-related mutations.
Insights
A novel fluorescent probe accurately detects KRAS mutations in cancer tissues, even at low levels. This highly specific and robust tool aids in personalized cancer therapy decisions.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- KRAS mutations are crucial biomarkers for guiding personalized cancer therapies.
- Accurate detection of these mutations in tumor tissues is essential for treatment selection.
Purpose of the Study:
- To develop and validate a novel fluorescent probe for sensitive and specific detection of KRAS mutations in cancer tissues.
- To assess the probe's performance under various conditions and in clinical samples.
Main Methods:
- Utilized a double-stranded toehold-exchange probe labeled with FAM (fluorescein amidite) and Dabcyl (4-(4-dimethylaminophenylazo)benzoic acid).
- Tested probe sensitivity down to 5% mutant alleles in a wild-type DNA background.
- Evaluated probe robustness across a range of temperatures (4°C–37°C) and salt concentrations (200 mM–1M Na+, 200 mM–500 mM K+).
- Validated the probe's clinical utility using 8 pairs of cancer and adjacent non-tumorous tissue samples, comparing results with direct sequencing.
Main Results:
- The probe demonstrated high specificity, differentiating target mutations in samples with as little as 5% mutant alleles.
- The probe maintained robust performance across diverse temperature and ionic strength conditions.
- Clinical validation showed complete agreement between probe detection results and direct sequencing.
Conclusions:
- The developed fluorescent probe offers extreme specificity and robustness for KRAS mutation detection.
- This probe represents a valuable tool for reliable molecular diagnosis of cancer-related mutations.
- Its performance supports its application in clinical settings for personalized genotype-based cancer therapy.
More Related Videos
10:57Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay
Published on: August 14, 2018
07:17Author Spotlight: Advancing the Detection of Low-Frequency Mutations in Cancer Tissues
Published on: August 23, 2024