FRET Cassette Signaling for Single-Channel Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Existing nucleic acid multiplex detection platforms require hardware changes or are technically challenging, limiting their adaptability to automated testing platforms and detection capacity.
Innovation Solution
A method utilizing invasive cleavage reactions with FRET cassettes and temperature-dependent quenching to distinguish different nucleic acid analytes in a single reaction mixture, allowing for detection using a single fluorescence channel without requiring hardware modifications.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If next generation sequencing technology is used for nucleic acid detection, then detection capacity and information acquisition are improved, but device complexity and technical challenge increase significantly
Solution Approach 1:
The patent replaces complex next generation sequencing technology with a simplified invasive cleavage assay system that uses fluorescent probes and temperature-dependent quenching mechanisms. This substitution maintains high detection capacity while dramatically reducing equipment complexity, enabling analysis on standard PCR instruments rather than requiring specialized sequencing equipment.
Solution Approach 2:
The patent utilizes temperature parameter changes to differentiate between multiple nucleic acid analytes. By adjusting reaction temperature to control quenching conditions, the system achieves multiplex detection without requiring complex hardware. This parameter-based differentiation allows a single fluorescence channel to distinguish between multiple targets through temperature-dependent signal characteristics.
2Productivity
If multiple fluorescent labels are used to detect different nucleic acid analytes, then detection capacity is improved, but device complexity increases due to requirement for multiple detection channels
Solution Approach 1:
The patent employs a single fluorescent label that serves multiple functions for detecting different nucleic acid analytes. Through temperature-dependent quenching mechanisms, the same fluorophore can be selectively activated or deactivated based on reaction conditions, allowing one detection channel to perform multiplex analysis that would traditionally require multiple specialized channels.
Solution Approach 2:
The system uses temperature as a control parameter to differentiate signals from multiple analytes detected with the same fluorescent label. By adjusting temperature to control quenching states, the single fluorescence channel can resolve multiple targets through temporal or conditional signal separation rather than requiring spectral separation.
3Measurement precision
If different probes produce different signals for multiple analytes, then analyte differentiation is improved, but signal interference and confidentiality issues arise
Solution Approach 1:
The patent employs temperature cycling or sequential temperature adjustments to periodically activate different analyte detections. By controlling temperature changes over time, the system can sequentially differentiate analytes without simultaneous signal generation, eliminating interference between different probe signals while maintaining clear differentiation capability.
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
Enhances detection capacity and adaptability to automated testing platforms by enabling multiplex nucleic acid analysis with efficient signal differentiation through temperature-dependent quenching mechanisms.
Implementation Method 1
The masking oligonucleotide includes a fluorescence quenching moiety attached thereto. When the hybridization between the masking oligonucleotide and the labeled cleaved flap occurs
Implementation Method 2
the quenching moiety of the masking oligo is brought into proximity of the fluorophore moiety of the cleaved 5' flap. Fluorescent signal emitted by the fluorophore of the cleaved 5' flap is then quenched
Implementation Method 3
it becomes possible to determine which FRET cassette, among a plurality of FRET cassettes labeled with fluorophores detectable in the same channel of a fluorometer, cleaved to generate the signal. This fluorescence quenching capacity, or temperature-dependent difference in fluorescent signal
Data Source
Figure 1
Figure 2A~2C
Figure 3
AI summary
A multiplexed nucleic acid amplification and detection system useful for detecting the presence of multiple specific nucleic acid sequences or single nucleotide polymorphisms (i.e., "SNPs") in a temperature-dependent fashion using only a single fluorescence detection channel of a nucleic acid analyzer. The technique can be carried out using standard PCR instrumentation equipped for fluorescence detection or monitoring.