Solid-Support Hydrolysis Probes for Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Current real-time PCR technologies face limitations in multiplexing capability due to the need for spectrally distinct fluorochromes for each assay, which increases costs and complexity, especially when detecting multiple target sequences.
Innovation Solution
The method involves using target-specific primers and probes attached to solid supports, where a nucleic acid polymerase with exonuclease activity cleaves the probes to release reporters, allowing for the detection of multiple targets using the same reporter dye by spatially distinguishing the probes on different solid supports.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spectrally distinct fluorochromes are used for each assay in multiplex PCR, then detection accuracy is improved, but device complexity and cost increase
Solution Approach 1:
The patent uses fluorescent reporters that emit light at different wavelengths (colors) when excited by a single laser wavelength. Different probe sequences are labeled with fluorophores having distinct emission spectra, allowing spectral differentiation of multiple targets. This enables multiplexed detection while using a single excitation source, reducing instrument complexity compared to requiring multiple lasers for each fluorochrome.
Solution Approach 2:
The invention employs a universal excitation approach where a single laser wavelength can excite multiple different fluorophore labels simultaneously. This multi-functional capability allows one light source to serve multiple detection channels, eliminating the need for separate excitation sources for each assay and thereby reducing device complexity while maintaining the ability to distinguish multiple targets spectrally.
2Measurement precision
If spectrally distinct fluorochromes are used for each assay in multiplex PCR, then detection accuracy is improved, but cost increases
Solution Approach 1:
The patent enables a single laser source to excite multiple fluorophore types simultaneously, creating a universal detection platform. This reduces the need for multiple expensive laser systems and associated optical components, thereby lowering instrument cost while maintaining the capability to perform multiple assays with spectrally distinct reporters for accurate detection.
Solution Approach 2:
By utilizing fluorophores with different emission colors that can all be excited by one laser wavelength, the system achieves multiplexed detection without requiring multiple expensive laser sources. The spectral differentiation of emitted colors provides detection accuracy while the single excitation source reduces overall system cost.
3Adaptability or versatility
If multiple fluorochromes are used for multiplex detection, then multiplexing capability is improved, but ease of operation worsens
Solution Approach 1:
The invention creates a universal excitation system where a single laser wavelength serves multiple fluorophore labels, simplifying the operational workflow. Users只需 set up one excitation source for all assays rather than configuring multiple lasers, reducing operational complexity while maintaining high multiplexing capability through spectral analysis of the emitted signals.
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 enhances multiplexing capability, reduces costs, and simplifies instrumentation by enabling the detection of multiple nucleic acids with a single reporter dye, improving the efficiency and accuracy of nucleic acid detection.
Implementation Method 1
cleaving the hybridized target-specific probe with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support
Implementation Method 2
contacting the sample with a first target-specific primer complementary to a first region on a first strand of the target nucleic acid, and a target-specific probe complementary to a second region on the first strand of the target nucleic acid downstream of the first region under conditions suitable for hybridization
Data Source
AI summary
Methods and compositions for the detection and quantification of nucleic acids are provided. In one embodiment, a sample is contacted with a primer complementary to a first region of a target nucleic acid and a probe complementary to a second region of the target nucleic acid downstream of the first region under conditions suitable for hybridization of the target nucleic acid with the primer and the probe. The probe in this embodiment comprises a fluorophore and is attached to a solid support. The hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the reporter from the solid support. The presence of the target nucleic acid is then detected and optionally quantified by detecting a decrease in signal from the reporter on the solid support.