Multiplexed Nucleic Acid Detection via Segmented Capture and Amplification
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Solution Overview
Problem
Conventional multiplexing technologies for nucleic acid analysis face limitations such as low sensitivity, cross-reactivity, and assay/instrumentation complexity, particularly in detecting low abundance target nucleic acids like microRNAs, mRNAs, and genomic DNAs.
Innovation Solution
The method involves contacting a sample with particles bearing capturing probes to capture and amplify target nucleic acids, followed by re-capturing with re-capturing probes, and detecting the labeled amplified nucleic acids using a detectable entity, allowing for the simultaneous analysis of multiple target nucleic acids in a single sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional multiplexing technologies are used for nucleic acid analysis, then multiple targets can be analyzed simultaneously, but sensitivity is reduced and cross-reactivity occurs
Solution Approach 1:
The patent divides the multiplexing process into two separate stages: (1) capture of target nucleic acids by immobilized probes on magnetic beads, and (2) detection of amplified products by soluble probes in solution. This segmentation allows each stage to be optimized independently, maintaining high sensitivity while enabling multiplexing. The capture stage uses stringent conditions to prevent cross-reactivity, while the detection stage benefits from signal amplification.
Solution Approach 2:
The patent extracts the amplification step and performs it in solution after capture, rather than attempting to amplify multiple targets simultaneously on the solid support. This extraction of the amplification function to a separate phase allows for more efficient signal generation and reduces cross-reactivity between multiple targets during the capture phase.
2Adaptability or versatility
If conventional multiplexing methods are employed, then multiple nucleic acid targets can be detected, but assay complexity increases
Solution Approach 1:
The patent employs universal amplification primers that can amplify multiple different target sequences simultaneously in a single PCR reaction. The capture probes are designed with a common adapter sequence that allows all captured targets to be amplified using the same primer set, significantly simplifying the assay compared to requiring separate amplification reactions for each target.
Solution Approach 2:
The patent combines multiple target capture events into a single solid-phase reaction, followed by a single amplification reaction in solution that generates detectable signals for all targets simultaneously. This merging of multiple operations into fewer steps reduces assay complexity while maintaining multiplexing capability.
3Measurement precision
If stringent capture conditions are used to prevent cross-reactivity, then specificity improves, but sensitivity to low abundance targets decreases
Solution Approach 1:
The patent performs target capture under stringent conditions first to ensure high specificity and prevent cross-reactivity. After capture is complete, the targets are then amplified under optimized conditions that maximize sensitivity. This preliminary action of capturing with high specificity before amplification allows the system to detect low abundance targets without cross-reactivity.
Solution Approach 2:
The patent changes the reaction parameters between the capture and detection phases. During capture, stringent hybridization conditions are used to ensure specificity. During the subsequent amplification and detection phase, parameters are optimized for signal generation and sensitivity, allowing detection of low abundance targets that were specifically captured in the first phase.
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 the sensitivity and reliability of nucleic acid analysis, enabling the detection of low abundance targets with high specificity and efficiency, as demonstrated by the ability to detect as few as 100 molecules per sample and providing robust microRNA profiling across different tissue types.
Implementation Method 1
contacting a sample with a first set of particles bearing a plurality of capturing probes, each comprising at least one target capturing sequence, under conditions that permit the capturing probes to capture one or more target nucleic acids in the sample
Implementation Method 2
amplifying the captured one or more target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the detectable entity
Implementation Method 3
incubating amplification product with a second set of particles bearing a plurality of re-capturing probes such that the amplified one or more target nucleic acids are re-captured by the plurality of the re-capturing probes
Data Source
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Figure 2A~2C
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AI summary
The present invention provides, among other things, methods of detecting target nucleic acid, comprising steps of: a) contacting a sample with one or more capturing probes, each comprising at least one target capturing sequence, under conditions that permit the one or more capturing probes to capture one or more target nucleic acids in the sample; b) amplifying the captured one or more target nucleic acids in a reaction mixture comprising a detectable entity such that the amplified one or more target nucleic acids are labeled with the detectable entity; c) incubating amplification product with a plurality of re-capturing probes such that the amplified one or more target nucleic acids are re-captured by the plurality of the re-capturing probes; and d) detecting signal generated by detectable entity associated with the re-captured amplified one or more target nucleic acids, wherein the presence and/or abundance of the detectable signal indicates the presence and/or abundance of the one or more target nucleic acids in the sample.