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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
Blocking probe as a potential tool for detection of single nucleotide DNA mutations: design and performance
M Ali Aboudzadeh1, M Sanromán-Iglesias, C H Lawrie
1Polymat, University of the Basque Country, 20018 Donostia-San Sebastián, Spain. thomas.schafer@ehu.es.
Abstract:
Developing strategies to detect single nucleotide DNA mutations associated with treatment decisions in cancer patients from liquid biopsies is a rapidly emerging area of personalized medicine that requires high specificity. Here we report how to design an easy enzyme-free approach that could create a platform for detection of L858R mutation of EGFR that is a predictive biomarker of tyrosine kinase treatment in many cancers. This approach includes the addition of blocking probes with the antisense ssDNA at different blocking positions and different concentrations such as to avoid re-annealing with the respective sense ssDNA. The successful blocking strategy was corroborated by fluorescence spectroscopy in solution using two distinct FRET pairs and quartz crystal microbalance with dissipation (QCM-D) measurements under comparable experimental conditions, as the hybridization rate-limiting step in both methods is the nucleation process. The efficiency of hybridization of each blocking probe was strongly dependent on its position particularly when the analyte possesses a secondary hairpin-structure. We tested the performance of blocking probes in combination with gold nanoparticles; the obtained results were in agreement with those of QCM-D. These findings could facilitate the development of better biosensors, especially those using probes containing secondary structure.
Insights
This study presents an enzyme-free biosensor for detecting the EGFR L858R mutation in cancer patients. The novel blocking probe strategy enhances specificity for personalized medicine applications.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Liquid biopsies are crucial for personalized cancer medicine, requiring highly specific detection of DNA mutations.
- EGFR L858R mutation is a key biomarker for tyrosine kinase inhibitor treatment in various cancers.
Purpose of the Study:
- To develop an enzyme-free biosensor platform for detecting the EGFR L858R mutation.
- To design an effective blocking probe strategy to enhance detection specificity.
Main Methods:
- Utilized blocking probes with antisense single-stranded DNA (ssDNA) at varied positions and concentrations.
- Employed fluorescence spectroscopy with Förster Resonance Energy Transfer (FRET) pairs and Quartz Crystal Microbalance with Dissipation (QCM-D) monitoring.
- Investigated probe performance with gold nanoparticles.
Main Results:
- Demonstrated successful blocking of non-target hybridization, confirmed by FRET and QCM-D.
- Showcased hybridization efficiency dependence on blocking probe position, especially for hairpin structures.
- Validated blocking probe efficacy in conjunction with gold nanoparticles, aligning with QCM-D data.
Conclusions:
- The developed enzyme-free approach offers a promising platform for EGFR L858R mutation detection.
- The blocking probe strategy significantly improves biosensor specificity, crucial for clinical applications.
- Findings support the development of advanced biosensors, particularly for targets with secondary structures.
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