Dark Quencher Fluorophore Probes for Nucleic Acid Detection
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Solution Overview
Problem
Current fluorescent oligonucleotide probes, particularly self-quenching probes, face challenges such as high background noise, complex design requirements due to the need for secondary structures, and high production costs, limiting their sensitivity, reliability, and quantitative detection capabilities in nucleic acid amplification and analysis.
Innovation Solution
Development of novel phosphoramidites and solid supports functionalized with stabilizing moieties and quenchers, such as 'Black Hole Quenchers', which facilitate efficient fluorescence energy transfer without requiring secondary structures, allowing for a broader range of nucleic acid sequences and improved multiplexing capabilities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If hairpin structures are used in fluorescent probes, then fluorescence quenching is achieved, but probe design becomes complex and hybridization is interfered with
Solution Approach 1:
The patent extracts the secondary structure requirement from the probe design by using dark quenchers that do not require hairpin or stem-loop structures. The quencher is simply incorporated into the oligonucleotide sequence, eliminating the need for complex structural design while maintaining quenching functionality.
Solution Approach 2:
The patent changes the quenching mechanism parameter from structure-dependent (hairpin) to sequence-dependent (dark quencher incorporation). This allows the quencher to function through its inherent dark properties rather than requiring specific structural configurations, simplifying probe design.
2Measurement precision
If conventional quenchers are used, then fluorescence detection is possible, but background noise remains high
Solution Approach 1:
The patent employs dark quenchers that are inherently non-fluorescent and do not require complex purification or activation. These quenchers provide immediate and consistent background reduction without the need for additional processing steps, effectively eliminating background noise from the detection system.
3Measurement precision
If fluorescent probes are designed for high sensitivity, then detection capability improves, but production cost increases
Solution Approach 1:
The patent changes the manufacturing parameter by using dark quenchers that can be directly incorporated into oligonucleotide synthesis without additional purification steps. This simplifies the production process and reduces costs while maintaining high detection sensitivity through the inherent dark properties of the quencher.
4Reliability
If secondary structures are required for probe function, then quenching mechanism is established, but probe versatility is limited
Solution Approach 1:
The patent extracts the secondary structure requirement from the system, allowing dark quenchers to function through their inherent properties rather than requiring hairpin or stem-loop structures. This enables the probes to target a broader range of nucleic acid sequences without being constrained by structural design requirements.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The new probes exhibit enhanced binding affinity and reduced background noise, enabling more sensitive, reliable, and quantitative detection of nucleic acids with simplified design and lower production costs.
Implementation Method 1
FET probes contain both a fluorophore and quencher tethered to an oligonucleotide. The fluorophore and the quencher are configured to produce a signal only as a result of hybridization to an intended target.
Implementation Method 2
Exemplary materials include a solid support and a phosphoramidite functionalized with a stabilizing moiety. Various materials are functionalized with both a stabilizing moiety and a quencher.
Data Source
AI summary
The present invention provides a new class of solids supports for synthesis of modified oligomers of nucleic acids, and nucleic acid probes that have a format expediently synthesized on the new supports. Exemplary solid supports include at least one quencher bound through a linker to the solid support. Various exemplary embodiments include a moiety that stabilizes a duplex, triplex or higher order aggregation (e.g., hybridization) of nucleic acids of which the oligomer of the invention is a component. Other components of the solid support include moieties that stabilize aggregations of nucleic acids, e.g., intercalators, minor groove binding moieties, bases modified with a stabilizing moiety (e.g., alkynyl moieties, and fluoroalkyl moieties), and conformational stabilizing moieties, such as those described in commonly owned U.S. Patent Application Publication No. 2007/0059752.