Modular PCR Reporter Complex for Flexible Multiplex DNA Detection
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Solution Overview
Problem
Current PCR detection methods using target sequence-specific DNA probes are complex, expensive, and inflexible due to the need for multiple biochemical labels and modifications, limiting multiplexing capabilities and precision.
Innovation Solution
A modular reporter complex composed of non-covalently linked oligonucleotides with labels, where a mediator probe is cleaved during PCR and binds to a target sequence-unspecific reporter complex, initiating a signal change without covalent bonds, allowing flexible design and efficient multiplex detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If target sequence-specific DNA probes with multiple biochemical labels and modifications are used, then detection precision is improved, but device complexity and manufacturing cost increase
Solution Approach 1:
The detection system is divided into two independent parts: a target sequence-specific mediator probe and a target sequence-unspecific reporter complex. The mediator probe contains the sequence-specific recognition element, while the reporter complex contains the fluorophore and quencher. This segmentation allows each component to be optimized independently, reducing overall complexity while maintaining detection precision.
Solution Approach 2:
A mediator probe acts as an intermediary between the target sequence and the reporter complex. The mediator probe binds to the target sequence and is subsequently cleaved by exonuclease activity, releasing a mediator that then binds to the reporter complex to generate the fluorescence signal. This intermediary mechanism decouples sequence specificity from signal generation, simplifying the overall system design.
2Measurement precision
If target sequence-specific DNA probes with multiple biochemical labels and modifications are used, then detection precision is improved, but manufacturing cost increases
Solution Approach 1:
By segmenting the detection system into a mediator probe and a reporter complex, the complex multi-labeled structure is divided into simpler components. The reporter complex with fluorophore and quencher can be synthesized separately from the sequence-specific mediator probe, reducing synthesis complexity and manufacturing cost while maintaining detection precision.
Solution Approach 2:
The reporter complex serves as a universal template that can be reused across different detection applications. Instead of synthesizing new multi-labeled probes for each target sequence, the same reporter complex structure can be paired with different mediator probes, significantly reducing manufacturing costs through standardized production.
3Measurement precision
If target sequence-specific DNA probes are used, then sequence-specific detection is achieved, but adaptability to different target sequences decreases
Solution Approach 1:
The detection system separates sequence-specific functions (mediator probe) from signal generation functions (reporter complex). This allows the reporter complex to be a universal component that can detect multiple different target sequences by pairing with different mediator probes, significantly improving adaptability while maintaining sequence-specific detection capability.
Solution Approach 2:
The reporter complex is designed as a universal detection molecule that can work with multiple different mediator probes targeting different sequences. This universal reporter complex approach enables a single probe design to serve multiple detection purposes across different target sequences, enhancing versatility and adaptability.
4Adaptability or versatility
If multiple DNA probes with different fluorophore-quencher combinations are used for multiplex detection, then multiplexing capability is improved, but device complexity increases
Solution Approach 1:
The mediator probe acts as an intermediary that carries sequence-specific information while the reporter complex generates the fluorescence signal. For multiplex detection, different mediator probes can be used with the same reporter complex structure, reducing complexity compared to using entirely different probe systems for each target. The intermediary mechanism allows standardized signal generation across multiple targets.
Solution Approach 2:
A universal reporter complex design can be used across multiple detection channels and target sequences. Instead of designing unique multi-labeled probes for each target, the same reporter complex structure serves multiple functions by pairing with different mediator probes, simplifying the overall multiplexing system while maintaining the ability to detect multiple targets simultaneously.
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
Enables precise and cost-effective multiplex detection of multiple DNA sequences in a single channel, overcoming limitations of current methods by providing a flexible and efficient PCR detection system with comparable performance to one-part detection molecules.
Implementation Method 1
These detection systems usually use light-absorbing and light-emitting fluorescent molecules. After excitation by light energy of a certain wavelength, these molecules emit energy in the form of higher wavelengths, which can be detected by detectors.
Implementation Method 2
A distinction is made between molecules whose light energy is to be detected in a specific wavelength range (fluorescence donor or fluorophore) and molecules that lead to a decrease in the fluorescence intensity of a fluorophore in close proximity (fluorescence acceptor or quencher). If the spatial proximity between fluorophore and quencher changes, the fluorescence signal changes accordingly, whereby a smaller distance always results in higher quenching (FRET quenching or contact quenching).
Data Source
AI summary
The invention relates to a method for detecting at least one target nucleic acid sequence by means of a mediator probe and at least one target sequence-unspecific modular reporter complex, wherein the released mediator sequence binds to a mediator binding site of the target sequence-unspecific modular reporter complex and is extended. A signal change is initiated and detected. The invention also relates to a kit for carrying out this method.