Multiplex Invasive Cleavage Assay Single Fluorophore Detection
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Solution Overview
Problem
Existing multiplex nucleic acid detection methods face challenges in independently detecting multiple target nucleic acids using a single detectable label, due to issues with oligonucleotide probe design, multiple label detection requirements, and compromised signal identification.
Innovation Solution
A method involving a multiplex invasive cleavage assay where multiple secondary reactions are permitted to occur under different temperature conditions, each specific to a unique target nucleic acid, and monitored for fluorescent signals, allowing for independent detection and distinction of multiple targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple detectable labels are used to detect multiple target nucleic acids, then detection capability is improved, but device complexity increases due to requiring multiple detection channels
Solution Approach 1:
A single detection channel is designed to perform multiple functions by detecting different target nucleic acids through temperature-dependent secondary reactions. The system uses one fluorophore and one detection channel to identify multiple targets by varying the temperature to activate different secondary reactions, making the detection system universal rather than requiring separate channels for each target.
Solution Approach 2:
The invention changes the temperature parameter to control which secondary reaction occurs. By adjusting the temperature, the system activates specific secondary reactions that are only permissive at certain temperature ranges, allowing a single detection channel to distinguish between multiple targets based on which temperature condition activates the corresponding secondary reaction.
2Device complexity
If a single detectable label is used for multiple targets, then device complexity is reduced, but measurement precision deteriorates due to compromised ability to identify the target responsible for generating the signal
Solution Approach 1:
Temperature-dependent secondary reactions serve as intermediaries between the single detectable label and the multiple target nucleic acids. Each secondary reaction is specific to a particular target and is activated only at certain temperature conditions. The secondary reaction acts as a mediator that links the single fluorophore signal to the specific target, enabling precise target identification despite using only one label.
Solution Approach 2:
The detection process is segmented into multiple temperature conditions, with each condition activating a specific secondary reaction for a particular target. By dividing the detection into discrete temperature steps, the system can attribute fluorescence signals to specific targets based on which temperature condition produced the signal, maintaining measurement precision while using a single label.
3Productivity
If multiple targets are detected simultaneously in a single reaction mixture, then productivity is improved, but difficulty of detecting and measuring increases due to signal overlap
Solution Approach 1:
The system uses periodic temperature cycling to activate different secondary reactions at different time points. By periodically changing the temperature through defined ranges, the system sequentially activates secondary reactions for different targets, allowing the detection system to measure each target's signal separately in time rather than having overlapping signals simultaneously.
Solution Approach 2:
The invention introduces dynamic temperature control to the reaction mixture, making the system adaptable rather than static. By dynamically adjusting the temperature to different conditions, the system can selectively activate different secondary reactions, enabling the detection of multiple targets in a single reaction mixture without signal overlap, as each target is detected under specific dynamic temperature conditions.
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 the independent detection and distinction of multiple target nucleic acids in a single reaction mixture using a single detectable label, overcoming previous limitations in multiplex nucleic acid analysis.
Implementation Method 1
The FRET cassette includes a donor fluorophore and an acceptor quencher. The donor fluorophore and the acceptor quencher are in energy transfer relationship with one another such that emission from the donor fluorophore is quenched when the donor fluorophore and the acceptor quencher are both attached to the same FRET cassette.
Implementation Method 2
The secondary reaction includes a flap endonuclease (FEN) enzyme. The FRET cassette includes a 5′ flap hybridizing sequence. The secondary reaction includes hybridizing the 5′ flap to the 5′ flap hybridizing sequence of the FRET cassette and cleaving the FRET cassette.
Data Source
AI summary
The present disclosure relates to compositions and methods, and related systems, products and kits, for performing temperature-dependent multiplex invasive cleavage assays, where a plurality of target nucleic acids are detected and distinguished from each other in a procedure using only a single fluorescent moiety as the reporter, and single channel fluorescence detection. In some embodiments, at least one of the plurality of target nucleic acids is an amplified nucleic acid, where progress in a thermal cycling amplification reaction is monitored as a function of time (i.e., real-time amplification).