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Depletion of wild-type target enhances the hybridization-based sensitivity of low-abundant mutation detection by reference capture probes
- Source :
- Sensors and Actuators B: Chemical
- Publication Year :
- 2022
- Publisher :
- ELSEVIER SCIENCE SA, 2022.
-
Abstract
- Nucleic acids duplex formation via hybridization is a crucial reaction in many processes and application across different disciplines. In life sciences the detection of mutations is an important application for which hybridization is used, e.g. in diagnostics via single-nucleotide variants (SNVs). This paper deals with the physicochemical aspects of hybridization-based detection of low-abundance mutations, which is challenging due to unavoidable competitive hybridization of high-abundant wild type sequence with the low-abundant variants. We apply two experimental methods based on theoretical hybridization models to show how sensing of DNA mutation can be significantly improved. This is implemented on two SNV biomarkers for which we first select a reference capture probe. This is a probe designed to match neither the wild type nor the SNV sequence, but to have an equal affinity to the wild-type as the SNV-matching probe. This allows the mutation-specific signal to be expressed as a ratiometric quantity, leading to increased assay robustness. Secondly, we selectively deplete the wild-type species by introducing an excess of wild-type-specific capture probes, and account for these depletion effects in the theoretical model. We demonstrate the detection of 0.05% mutant species in a wild-type background, which is an improvement of an order of magnitude in the limit of detection in comparison with the nodepletion case. This sensitivity is comparable with digital PCR results, showing performance suitable for e.g. clinical applications in liquid biopsy context. The principles of this work apply to a wide range of hybridizationbased DNA biosensing technologies, irrespective of the underlying transducer principle. This work was supported by the Research Foundation Flanders (FWO) and the Flemish Institute for Technological Research VITO nv (grant numbers 1S69320N, 1159719N ) and by the EU H2020 M3DLoC project (grant number 760662).
- Subjects :
- Technology
EGFR
LoD, limit of detection
sensors
Microarray technology
T, target
PCR, polymerase chain reaction
S, signal
Materials Chemistry
KRAS, Kirsten rat sarcoma viral oncogene homolog
Electrochemistry
ctDNA, circulating tumor DNA
Electrical and Electronic Engineering
dPCR, digital PCR
Instrumentation
Instruments & Instrumentation
SNV, single-nucleotide variant
Science & Technology
Ref, reference
Reference capture probes
I, intensity
Target depletion
Chemistry, Analytical
Metals and Alloys
Mut, mutant
Hybridization-based nucleic acid mutation sensors DNA thermodynamics Target depletion Single nucleotide variant Microarray technology Reference capture probes Abbreviations: COSMIC, Catalog, ue Of Somatic Mutations In Cancer
NGS, next generation sequencing
P, probe
DNA
Condensed Matter Physics
S 0 , baseline signal
WT, wild-type
LRG, Locus Reference Genomic
Surfaces, Coatings and Films
Electronic, Optical and Magnetic Materials
EGFR, epidermal growth factor receptor
Hybridization-based nucleic acid mutation
Single nucleotide variant
Chemistry
PCR-METHOD
Physical Sciences
DNA thermodynamics
Subjects
Details
- Language :
- English
- Database :
- OpenAIRE
- Journal :
- Sensors and Actuators B: Chemical
- Accession number :
- edsair.doi.dedup.....4dad1bad7d75a270864d151971abdf89