Melt-Profile Cleavable Probes for Single-Fluorophore Multiplexing
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Solution Overview
Problem
Current real-time PCR technologies are limited in multiplexing capability due to the need for spectrally distinct fluorochromes and require multiple emission sources and detectors, increasing costs and complexity.
Innovation Solution
The use of cleavable probes comprising non-natural nucleotides labeled with reporter-quencher pairs and ribonucleotides, which undergo hybridization, cleavage, and extension to form hairpin probes, allowing for increased multiplexing through distinct melt profiles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If spectrally distinct fluorochromes are used for each assay in multiplex reactions, then detection of multiple target sequences is enabled, but instrument complexity and cost increase due to requiring multiple emission sources, detectors, and filters
Solution Approach 1:
The patent changes the detection parameter from spectral differentiation to thermal differentiation. By designing probes with distinct melting temperatures (Tm) that differ by at least 2°C, multiple targets can be detected using a single fluorophore, eliminating the need for multiple emission sources, detectors, and filters while maintaining multiplexing capability
Solution Approach 2:
The patent makes a single fluorophore system universal for detecting multiple targets by using probes with different Tm values. This allows one instrument configuration to detect multiple target sequences simultaneously through melt curve analysis, rather than requiring separate detection systems for each target
2Adaptability or versatility
If spectrally distinct fluorochromes are used for each assay in multiplex reactions, then detection of multiple target sequences is enabled, but cost increases due to requiring multiple emission sources, detectors, and filters
Solution Approach 1:
The patent changes the detection parameter from spectral differentiation to thermal differentiation. By designing probes with distinct melting temperatures (Tm) that differ by at least 2°C, multiple targets can be detected using a single fluorophore, eliminating the need for multiple emission sources, detectors, and filters while maintaining multiplexing capability
Solution Approach 2:
The patent uses inexpensive probe designs with distinct Tm values that can be synthesized with standard nucleotides. These probes are consumed in the reaction but provide cost-effective multiplexing compared to expensive multi-channel instrumentation
3Adaptability or versatility
If cleavable probes with ribonucleotides are used, then multiplexing capability is enhanced through distinct melt profiles, but probe design and synthesis complexity increases
Solution Approach 1:
The patent segments the probe into distinct functional regions: a 5' region containing the fluorophore and Tm-modifying elements, a central region with ribonucleotides for cleavage, and a 3' region for target hybridization. This segmentation allows systematic design of probes with predictable Tm values and cleavage properties
Solution Approach 2:
The patent changes the detection parameter from spectral differentiation to thermal differentiation. By designing probes with distinct melting temperatures (Tm) that differ by at least 2°C, multiple targets can be detected using a single fluorophore, eliminating the need for multiple emission sources, detectors, and filters while maintaining multiplexing 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
This approach significantly enhances multiplexing capabilities, enabling the detection of multiple target sequences with a single fluorophore by utilizing unique melt profiles, reducing instrument complexity and cost.
Implementation Method 1
contacting the cleavable probe with an endoribonuclease, thereby cleaving probe that is hybridized with target nucleic acid
Implementation Method 2
allowing the truncated cleavable probe to hybridize to itself to form a hairpin probe
Implementation Method 3
extending the hairpin probe
Implementation Method 4
detecting the target nucleic acid by detecting a change in signal from the label
Implementation Method 5
performing a melt analysis on the hairpin probe
Data Source
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AI summary
Methods and compositions for the detection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of cleavable probes that comprise a ribonucleotide position that is susceptible to endoribonuclease (e.g., RNase H) cleavage in the presence of target nucleic acid molecules. Probes of the embodiments may also comprise non-natural nucleotide linked to a reporter and/or quenching moiety.