Multiplexed Hydrolysis Probe Assay for 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, leading to increased costs and complexity, especially when detecting multiple target sequences.
Innovation Solution
A method involving target-specific primers and probes with quenchers, where the quencher is cleaved by exonuclease activity and the remaining probe hybridizes to a reporter probe attached to a solid support, allowing for detection of nucleic acids through signal changes, enabling multiplexing without the need for multiple fluorochromes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If spectrally distinct fluorochromes are used for each assay in a multiplex reaction, then detection of multiple target sequences is enabled, but instrument complexity and cost increase due to requiring multiple lasers and filters
Solution Approach 1:
A single fluorophore (e.g., FAM) is used across multiple assays in the multiplex reaction, with each assay distinguished by a unique probe sequence rather than requiring a different fluorophore. This universal fluorophore approach allows the same detection channel to detect multiple targets, eliminating the need for multiple lasers and filters while maintaining multiplexing capability
Solution Approach 2:
The invention introduces a probe as an intermediary element that provides assay-specific identification through its sequence rather than through spectral properties. The probe hybridizes to the target and brings the fluorophore into proximity with the target sequence, enabling specific detection without requiring the fluorophore itself to be assay-specific
2Adaptability or versatility
If spectrally distinct fluorochromes are used for each assay in a multiplex reaction, then detection of multiple target sequences is enabled, but cost increases due to requiring multiple lasers and filters
Solution Approach 1:
A single fluorophore (e.g., FAM) is used across multiple assays in the multiplex reaction, with each assay distinguished by a unique probe sequence rather than requiring a different fluorophore. This universal fluorophore approach allows the same detection channel to detect multiple targets, eliminating the need for multiple lasers and filters while maintaining multiplexing capability
Solution Approach 2:
The invention uses the same fluorophore 'copy' (e.g., multiple FAM-labeled probes) for different assays, relying on sequence specificity rather than spectral differentiation. This allows replication of the successful single-plex assay design across multiple targets without the need to purchase and implement multiple different fluorophore systems
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 allowing for the detection of multiple target sequences using a single fluorophore, while maintaining sensitivity and precision.
Implementation Method 1
cleaving the hybridized target-specific probe with a nucleic acid polymerase having exonuclease activity to release the quencher from the target-specific probe
Implementation Method 2
hybridizing any remaining target-specific probe to a reporter probe that is complementary to the target-specific probe, said reporter probe comprising a reporter and being attached to a solid support
Implementation Method 3
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 under conditions suitable for hybridization
Implementation Method 4
detecting the target nucleic acid by detecting a change in signal from the reporter in association with the solid support
Data Source
Figure 1~2
Figure 3~4
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 and a quencher-probe complementary to a target nucleic acid. The quencher-probe is complementary to an anti-probe that comprises a reporter and is attached to a solid support. Thus, hybridized probe is cleaved with a nucleic acid polymerase having exonuclease activity to release the quencher from the probe. The presence of the target nucleic acid is then detected and/or optionally quantified by detecting an increase in signal from the fluorescent reporter on the solid support.