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Related Experiment Video

Updated: Mar 26, 2026

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions
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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.

Biosensors & Bioelectronics
|February 2, 2016
PubMed
Summary

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.

Keywords:
DNAKRASMutationToehold-exchange Probe

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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.