Multiplexed Real-Time PCR Using Temperature-Corrected Fluorescence Signals
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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 restriction of spectrally-resolvable fluorophores, allowing for detection of only up to seven separate fluorescently labeled probes in the same tube, which is insufficient for clinical needs.
Innovation Solution
The use of novel oligonucleotide probes with a non-complementary tag portion and a quenching molecule, along with a dual-labeling system where the reporter moiety is separated from the quencher moiety by a nuclease susceptible cleavage site, allows for the detection of multiple targets by measuring temperature-corrected signals and calculating signal values across multiple PCR cycles.
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 bandwidth limitation restricts detection to only about six or seven dyes without significant overlap interference
Solution Approach 1:
The patent changes the detection parameter from spectral wavelength discrimination to temperature-dependent fluorescence signal discrimination. By using a single fluorophore with temperature-sensitive quenching characteristics, the system can distinguish multiple targets based on their different melting temperatures rather than requiring multiple spectrally distinct fluorophores. This parameter change enables detection of many more targets than the spectral bandwidth would normally permit.
2Measurement precision
If fluorescent dyes are used for probe labeling, then target 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 patent transitions from one-dimensional spectral discrimination (wavelength) to two-dimensional discrimination by adding temperature as a second dimension. The fluorescence signal intensity becomes a function of both wavelength (single fluorophore emission spectrum) and temperature (temperature-dependent quenching), allowing multiplexed detection of multiple targets that would otherwise be indistinguishable by spectrum alone.
3Quantity of substance
If multiple probes with distinct fluorescent labels are used in the same reaction, then multiple targets can be detected, but the detection instrument must be able to discriminate among the light signals emitted by each probe
Solution Approach 1:
The patent makes the single fluorophore system universal for detecting multiple targets by exploiting the temperature-dependent quenching property. The same fluorophore can detect different targets by measuring fluorescence at different temperatures, eliminating the need for instruments with multiple detection channels and complex spectral unmixing capabilities.
4Measurement precision
If the reporter moiety is separated from the quencher moiety by a nuclease susceptible cleavage site, then probe hydrolysis enables detection, but the quenching efficiency must be maintained until cleavage occurs
Solution Approach 1:
The patent creates a dynamic quenching system where the quenching efficiency changes with temperature. At lower temperatures, the quencher is close to the fluorophore providing strong quenching. As temperature increases during PCR cycling, the quencher moves away or its quenching efficiency decreases, allowing fluorescence signal to increase as the probe is hydrolyzed by nucleases. This dynamic behavior enables both stable baseline quenching and sensitive detection of hydrolysis events.
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 the detection and quantification of multiple nucleic acid targets in a single reaction, overcoming the limitations of existing methods and enhancing the ability to analyze numerous targets in real-time PCR assays.
Implementation Method 1
The reporter moiety is separated from the first quencher moiety by a nuclease susceptible cleavage site
Implementation Method 2
a quenching molecule, along with a dual-labeling system where the reporter moiety is separated from the quencher moiety
Implementation Method 3
the nuclease activity of the nucleic acid polymerase allows cleavage and separation of the tag portion from the first quencher moiety
Implementation Method 4
amplifying the target nucleic acid in the reaction vessel containing the mixture by PCR using a nucleic acid polymerase having 5′ to 3′ nuclease activity
Implementation Method 5
the annealing portion comprises a nucleotide sequence at least partially complementary to the target nucleic acid sequence and which hybridizes to a region of the target nucleic acid
Implementation Method 6
the tag portion hybridizes to a quenching oligonucleotide that comprises one or more quencher moieties capable of quenching the reporter moiety when the quenching oligonucleotide is hybridized to the tag portion
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 TAGS hydrolysis probes.