Probe Sets for Melt-Based Multiplex Detection in Nucleic Acid Assays
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Solution Overview
Problem
Current real-time PCR technologies have limited multiplexing capabilities due to the need for spectrally distinct fluorochromes and require multiple emission sources and detectors, increasing costs and limiting the number of targets that can be detected in a single reaction.
Innovation Solution
The use of probe sets and primer sets that form T-junctions or hairpin structures with non-natural nucleotides and reporter-quencher pairs allows for the detection of multiple targets by distinguishing signal changes based on melt points, enabling higher multiplexing without the need for multiple fluorochromes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spectrally distinct fluorochromes are used for each assay in multiplex PCR, then detection capability is improved, but device complexity and cost increase due to multiple emission sources and detectors
Solution Approach 1:
The invention changes the detection parameter from spectral differentiation to thermal differentiation. By using probes with distinct melting temperatures (Tm) rather than distinct fluorochrome emission spectra, the system can distinguish multiple targets using a single fluorochrome and single detector, eliminating the need for multiple emission sources and detectors while maintaining multiplex detection capability
Solution Approach 2:
The invention makes a single fluorochrome and single detector system universal for detecting multiple targets. By designing probes with different Tm values that all use the same fluorochrome-reporter system, one instrument configuration can perform multiplex detection of multiple targets, making the detection system universally applicable to various assays without requiring additional hardware
2Adaptability or versatility
If multiple emission sources and detectors are used for spectral differentiation, then multiplexing capability is improved, but cost increases
Solution Approach 1:
The invention changes the differentiation parameter from spectral properties to thermal properties (melting temperature). This allows multiple probes to be detected using the same fluorochrome and detector, eliminating the need for expensive multiple emission sources and detectors while maintaining the ability to distinguish multiple targets through their distinct Tm values
Solution Approach 2:
The invention uses the same fluorochrome-reporter probe design template for multiple targets, copying the successful single-plex assay design. By creating multiple copies of the probe structure with modified sequences that give different Tm values but identical fluorochrome labeling, the system achieves multiplexing without additional hardware costs
3Ease of operation
If free floating fluorochromes are used, then detection simplicity is maintained, but multiplexing capability is limited to 1-6 plex
Solution Approach 1:
The invention changes the detection parameter from spectral wavelength to melting temperature. By using probes with distinct Tm values instead of distinct fluorochrome emission spectra, the system can distinguish more targets without increasing spectral complexity, maintaining ease of operation while significantly enhancing multiplexing capability beyond the traditional 1-6 plex limit
Solution Approach 2:
The invention adds a thermal dimension to the detection space. Instead of relying solely on spectral differentiation in the optical domain, the system introduces temperature as an additional dimension for target differentiation, allowing probes to be distinguished by their melting behavior rather than their emission wavelengths
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 the multiplexing capacity of real-time PCR, allowing for the detection of multiple nucleic acid sequences in a single reaction, reducing costs and instrument complexity.
Implementation Method 1
at least one non-natural nucleotide labeled with a first member of a reporter-quencher pair
Implementation Method 2
when hybridized to the target nucleic acid the set of probes form a T-junction
Implementation Method 3
distinguishing signal changes based on melt points
Data Source
Figure 1A~1D
Figure 1E~1F
Figure 2A~2D
AI summary
Methods and compositions for the détection and quantification of nucleic acids are provided. In certain embodiments, methods involve the use of primers or probes that comprise a non-natural nucléotide linked to a reporter. Target nucleic acids are detected by the polymerization of a complementary probe or primer that incorporated a cognate non-natural nucléotide linked to a quencher.