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A genosensor for detecting single-point mutations in dendron chips after blocked recombinase polymerase amplification
Sara Martorell1,2, Ángel Maquieira1,2,3, Luis A Tortajada-Genaro1,2,3
1Instituto Interuniversitario de Investigación de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Universitat Politècnica de València, Universitat de València, Camino de Vera s/n, 46022 Valencia, Spain. luitorge@qim.upv.es.
The Analyst
|April 20, 2022
Summary
This study introduces a novel biosensing system for precise single nucleotide change detection. The method uses dendron crosslinkers and isothermal amplification for sensitive DNA variant genotyping.
Area of Science:
- Biotechnology
- Molecular Biology
- Genomics
Background:
- Accurate detection of single nucleotide variants is crucial for genetic diagnostics and personalized medicine.
- Existing microarray technologies face challenges in sensitivity and specificity for certain genetic variations.
Purpose of the Study:
- To develop a novel biosensing system for sensitive and specific detection of single nucleotide changes.
- To integrate isothermal amplification with a dendron-mediated DNA hybridization assay for enhanced genotyping.
- To validate the system's performance using clinical cancer samples.
Main Methods:
- Development of allele-specific probes using oligonucleotide-dendron conjugates via click chemistry (thiol-yne reaction).
- Immobilization of probes onto photo-activated polycarbonate substrates using carbodiimide reaction, creating forest-array chips.
- Utilizing blocked recombinase polymerase amplification (blocked RPA) at 37 °C for target DNA amplification.
- Hybridization of amplified products with immobilized probes for nucleotide genotyping and colorimetric detection.
Main Results:
- The developed dendron-mediated assay demonstrated 10-100 fold higher sensitivity compared to linear crosslinkers.
- Excellent analytical performance was achieved in both planar chip and fluidic formats.
- The system successfully identified the H1047R mutation in the PIK3CA gene from human cancer tissues.
- High accuracy (>95%) and a low limit of detection (0.02 ng μL⁻¹ genomic DNA) were achieved.
Conclusions:
- The novel biosensing system offers a reliable, versatile, and cost-effective method for sensitive and specific DNA variant detection.
- The integration of dendron crosslinkers significantly enhances hybridization assay performance.
- This technology shows promise for clinical applications in genetic mutation detection, including cancer diagnostics.
