Multiplexed Real-Time PCR Using Temperature-Dependent Probe Melting
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Solution Overview
Problem
Current real-time PCR methods are limited in their ability to simultaneously detect and quantify multiple nucleic acid targets due to the constraints of spectrally-resolvable fluorophores, allowing for detection of only four to seven separate targets in a single reaction vessel, which is insufficient for modern clinical needs.
Innovation Solution
The use of novel oligonucleotide probes with a non-complementary tag portion and a quenching molecule, where the tag portion is reversibly bound to a quenching molecule that quenches a reporter moiety, allowing for temperature-dependent signal measurement and calculation to detect multiple targets in a single reaction vessel.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple fluorescently-labeled probes are used to detect multiple targets, then the number of detectable targets increases, but the spectral overlap between fluorophores limits detection to only four to seven targets
Solution Approach 1:
The invention changes the detection parameter from spectral wavelength to temperature. By using a single fluorophore (e.g., FAM) for all probes and distinguishing targets based on their melting temperatures (Tm), the system can detect up to 21 targets without spectral overlap limitations. Each probe-target duplex has a unique Tm that serves as its identifier.
Solution Approach 2:
The invention segments the detection process into distinct temperature steps. After PCR amplification, the system performs a melting curve analysis where temperature is gradually increased, and fluorescence signals are measured at multiple temperature points. This temporal and thermal segmentation allows resolution of multiple targets with a single fluorophore.
2Adaptability or versatility
If more than four to seven targets are detected in a single reaction vessel, then clinical diagnostic capability improves, but current detection instruments cannot discriminate among light signals from more than four to seven separate wavelengths
Solution Approach 1:
The invention introduces temperature as an intermediary parameter to mediate between the fluorophore signal and target identification. Instead of directly measuring spectral differences, the system uses temperature-dependent melting behavior as an intermediary to distinguish targets. The melting curve (fluorescence vs. temperature) serves as the intermediary that encodes target identity.
Solution Approach 2:
The invention adds the temperature dimension to the detection space. Rather than relying solely on the wavelength dimension (which is limited to 4-7 distinguishable channels), the system utilizes the temperature dimension by measuring fluorescence at multiple temperature points during a melting curve analysis, effectively creating a two-dimensional detection space (wavelength × temperature) that can resolve many more targets.
3Measurement precision
If fluorescent dyes are used for probe labeling, then real-time detection is enabled, but only about six or seven dyes can be fit within the visible spectrum without significant overlap interference
Solution Approach 1:
The invention makes a single fluorophore (e.g., FAM) universal for detecting all targets in the multiplex assay. Instead of assigning different fluorophores to different targets, the same fluorophore is used for all probes, and its function is extended to differentiate targets through temperature-dependent melting behavior. This universal labeling approach eliminates the need for multiple spectrally-distinct dyes.
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 detection and quantification of multiple nucleic acid targets in a single reaction vessel, overcoming the limitations of existing methods by allowing for the analysis of numerous targets beyond the conventional four to seven, enhancing clinical applications such as viral load determination and tumor molecular signature analysis.
Implementation Method 1
the tag portion is reversibly bound in a temperature-dependent manner to a quenching molecule that comprises or is associated with one or more quencher moieties capable of quenching the reporter moiety when the quenching molecule is bound to the tag portion
Implementation Method 2
a quenching molecule that comprises or is associated with one or more quencher moieties capable of quenching the reporter moiety
Implementation Method 3
amplifying the target nucleic acid by PCR using a nucleic acid polymerase having 5′ to 3′ nuclease activity such that during an extension step of each PCR cycle, the nuclease activity of the polymerase allows cleavage and separation of the reporter moiety from the first quencher moiety
Implementation Method 4
measuring a suppressed signal from the reporter moiety at a first temperature at which the quenching molecule is bound to the tag portion; increasing temperature to a second temperature at which the quenching molecule is not bound to the tag portion; measuring a temperature corrected signal from the reporter moiety at the second temperature
Data Source
AI summary
The present invention describes methods for performing higher multiplexed real-time PCR for detection and quantitation of target nucleic acids using tagged hydrolysis probes.