Intra-Channel Nucleic Acid Multiplexing via Thermal Signal Separation
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Solution Overview
Problem
Existing multiplex nucleic acid detection assays face challenges due to substantial overlap in emission spectra of detectable labels, limiting the number of targets that can be detected and measured, and requiring complex deconvolution algorithms to resolve separate fluorescence signals.
Innovation Solution
The use of cleavable and non-cleavable probes, such as extendable fluorogenic probes, within the same detection channel, combined with a three-stage thermal cycling method, allows for the differentiation of signals from different targets by altering the emission spectra through specific interactions with nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple detectable labels with different emission spectra are used to detect multiple target nucleic acids, then the number of detectable targets increases, but the emission spectra overlap substantially making signal resolution difficult
Solution Approach 1:
The patent applies dynamics by using thermal cycling to dynamically change the binding states of probes. During denaturation, all probes release from targets; during annealing, probes rebind specifically. This dynamic state change allows sequential measurement of different targets at different cycle phases, resolving spectral overlap issues.
Solution Approach 2:
The patent implements periodic action through thermal cycling with repeated denaturation and annealing phases. Each cycle allows probes to bind and release periodically, enabling time-resolved detection where signals from different targets are measured at different periodic phases, thus resolving spectral overlap.
2Adaptability or versatility
If detectable labels with overlapping emission spectra are used, then more targets can be detected in a single reaction mixture, but complex deconvolution algorithms are required to resolve separate fluorescence signals
Solution Approach 1:
The patent applies preliminary action by pre-designing probes with different binding kinetics and stability. Some probes are designed to bind tightly and remain bound during denaturation, while others release completely. This preliminary differentiation of binding characteristics allows direct signal measurement without complex deconvolution algorithms.
Solution Approach 2:
The patent uses dynamics to create time-separated signal detection. By controlling thermal cycling conditions, signals from different targets are generated at different times during the cycle, allowing direct measurement without mathematical deconvolution of overlapping spectra.
3Device complexity
If the same detection channel is used for multiple targets, then the assay complexity is reduced, but the signals from different targets cannot be distinguished
Solution Approach 1:
The patent introduces a temporal dimension to detection by using thermal cycling phases. Instead of relying solely on spectral differentiation in the wavelength dimension, the invention measures signals at different time points corresponding to different thermal phases, effectively adding a time dimension to resolve target-specific signals in the same detection channel.
Solution Approach 2:
The patent applies dynamics by exploiting the different binding behaviors of probes during thermal cycling. Some probes dynamically bind and release at different temperatures, allowing the same detection channel to measure different targets at different dynamic states, thereby maintaining measurement precision without increasing channel complexity.
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 enables robust multiplex nucleic acid detection by effectively resolving signals from targets with overlapping emission spectra, increasing the number of targets that can be detected in a single reaction mixture without complex deconvolution, and improving the accuracy of quantification.
Implementation Method 1
the detectable labels are fluorescent dyes integrated with a nucleic acid probe, a primer, or some other nucleic acid molecule designed to specifically hybridize with the corresponding target nucleic acid
Implementation Method 2
a three-stage thermal cycling method, allows for the differentiation of signals from different targets by altering the emission spectra through specific interactions with nucleic acids
Data Source
AI summary
Disclosed are compositions, kits, and methods that enable intra-channel multiplexing by enabling determination of separate detectable signals, each associated with a different assay target, within the same detection channel. The multiple detectable signals can be separately resolved and independently analyzed to enable detection and/or quantification of each respective target. Enabling multiple targets to be assayed within the same detection channel increases the plexy of multiplex assays without relying on additional dyes and concomitant issues of increased spectral overlap.