Nucleic Acid Detection Using Fluorescence Quenching
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Solution Overview
Problem
Conventional methods for detecting nucleic acids are often insensitive, time-consuming, and costly, requiring elaborate detection methods and expensive reagents, with limitations in sensitivity and efficiency, especially at low concentrations.
Innovation Solution
A method involving the use of a dye and an absorber in a solution, where the absorber's binding to the nucleic acid attenuates both excitation and emission light independently of the dye's quantum yield, allowing for efficient detection without the need for intercalating dyes or specific spatial arrangements, and can be used in conjunction with conventional amplification and detection devices.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional intercalating dyes (e.g., Sybr-Green) are used for nucleic acid detection, then the fluorescence signal increases upon binding to double-stranded DNA, but the detection limit is relatively high (around 10 nM) and the amplification reaction duration becomes very long
Solution Approach 1:
The patent changes the detection parameter from measuring fluorescence intensity increase (conventional method) to measuring fluorescence quenching attenuation. By using a quencher that binds to the nucleic acid and attenuates the dye's fluorescence, the detection becomes sensitive at much lower concentrations (attomolar range), thereby reducing the amplification reaction duration while maintaining high detection sensitivity.
2Measurement precision
If conventional dye-based detection methods are used, then detection can be performed during amplification, but the detectable limit is attained only at relatively high concentrations requiring many amplification cycles
Solution Approach 1:
The patent introduces a quencher as an intermediary substance that binds to the nucleic acid and attenuates the fluorescence signal. This intermediary mechanism enables detection at much lower concentrations by creating a more sensitive signal change, allowing faster detection and reducing the number of amplification cycles needed to reach the detection threshold.
3Measurement precision
If FRET-based detection methods (e.g., TaqMan probes) are used, then specific nucleic acid sequences can be detected, but the method requires specific spatial arrangements and more complex probe designs
Solution Approach 1:
The patent extracts the quencher from the probe structure itself (as in FRET-based methods where quencher is part of the probe) and makes it a separate component that binds to the nucleic acid. This simplification eliminates the need for complex probe designs with built-in quenchers and specific spatial arrangements, reducing method complexity while maintaining sequence detection capability through the separate quencher binding mechanism.
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
This approach significantly reduces the time to obtain meaningful results, enhances sensitivity, and allows for cost-effective detection of nucleic acids, even at low concentrations, without the need for expensive equipment or complex spatial arrangements, and can be integrated with existing PCR methods.
Implementation Method 1
the at least one dye is adapted to emit emission light due to an optical excitation by excitation light
Implementation Method 2
the absorber is adapted to cause an attenuation of the emission light and/or the excitation light
Data Source
Figure 1A~1C
Figure 1D
Figure 2A~2B
AI summary
The invention relates to a method for detecting at least one nucleic acid to be detected (10) in a solution, comprising the steps of: providing at least one dye (8), wherein the solution and/or a reaction vessel (R), wherein the solution is present, comprises the dye (8) and wherein the at least one dye (8) is adapted to emit emission light due to an optical excitation by excitation light; providing at least one absorber (1) in the solution, wherein the absorber (1) is adapted to cause an attenuation of the emission light and/or the excitation light and wherein the attenuation is influenced by a bonding of the at least one absorber (1) to the nucleic acid to be detected (10); and radiating excitation light into the solution and measuring an intensity of the emission light; wherein the attenuation of the emission light and/or the excitation light by the at least one absorber occurs independently of a quantum yield of the at least one dye. Furthermore, the present invention relates to a corresponding kit, comprising at least one corresponding dye and at least one corresponding absorber.