PTO Cleavage and Extension Oligonucleotide Hybridization for Nucleic Acid Detection
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Solution Overview
Problem
Conventional methods for detecting target nucleic acid sequences on a solid phase suffer from false positives due to non-specific hybridization and require multiple fluorescent labels, limiting the number of sequences that can be detected and complicating reaction optimization.
Innovation Solution
The PCE-IH assay employs a PTO Cleavage and Extension-Dependent Immobilized Oligonucleotide Hybridization method, where a PTO is hybridized with a target sequence, cleaved by a 5' nuclease enzyme, and the released fragment is extended and hybridized with a CTO, forming an extended duplex that is immobilized on a solid substrate, allowing for accurate detection with a single label.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional probe hybridization methods are used on a solid phase, then detection of target sequences can be performed, but false positive results occur due to non-specific hybridization
Solution Approach 1:
The probe is divided into multiple functional domains: a target-binding domain that specifically recognizes the target sequence, a spacer domain that provides physical separation, and a reporter domain that generates the detectable signal. This segmentation allows the target-binding domain to maintain high specificity while the spacer domain prevents non-specific interactions with the solid phase surface, thereby reducing false positives without compromising detection accuracy
Solution Approach 2:
A spacer domain acts as an intermediary element between the target-binding domain and the solid phase surface. This spacer domain physically separates the probe from the surface, preventing non-specific adsorption and hybridization events while allowing the target-binding domain to freely access and bind to complementary target sequences in solution, thus eliminating false positives caused by surface-induced non-specific binding
2Adaptability or versatility
If multiple fluorescent labels are used to detect multiple target sequences, then the number of detectable sequences increases, but the system complexity and reaction optimization difficulty increase
Solution Approach 1:
A single fluorescent label attached to the probe performs multiple functions: it serves as the detection reporter and simultaneously enables differentiation of multiple target sequences through melting temperature analysis. The label is positioned in a spacer domain that provides universal functionality across different probe designs, allowing the same labeling strategy to be applied universally while detecting multiple distinct targets through thermal melting profiles rather than requiring multiple different labels
Solution Approach 2:
The system utilizes changes in melting temperature (Tm) as a parameter to differentiate between multiple target sequences. Each probe-target hybridization produces a characteristic Tm value that serves as a unique identifier for the specific target sequence. By monitoring Tm shifts rather than relying on multiple fluorescent emission wavelengths, the system achieves multiplex detection capability while maintaining a simple single-label configuration, thereby reducing system complexity while preserving versatility
3Device complexity
If a single fluorescent label is used for detection, then system simplicity is maintained, but the ability to detect multiple target sequences is limited
Solution Approach 1:
The system exploits parameter changes in melting temperature (Tm) to enable multiplex detection with a single fluorescent label. Each probe-target hybridization complex exhibits a distinct Tm value characteristic of its specific sequence match. By monitoring the Tm of the fluorescently-labeled probe, the system can distinguish between multiple different target sequences bound to different probes on the solid phase, thus achieving versatility while maintaining the simplicity of a single-label system
Solution Approach 2:
The system transitions from detecting multiple targets through multiple spatial dimensions (different fluorescent wavelengths) to detecting them through a temporal/thermal dimension (melting temperature). By using Tm as an additional dimension of discrimination, the system can resolve multiple target sequences using a single fluorescent label, effectively adding a new dimension of detection capability without increasing the number of labels or system complexity
4Measurement precision
If probes are immobilized on a solid phase, then detection sensitivity is improved, but non-specific hybridization increases causing false positives
Solution Approach 1:
The spacer domain serves as an intermediary that physically separates the probe from the solid phase surface. This separation prevents direct contact between the probe and surface, eliminating non-specific adsorption and hybridization events that cause false positives. At the same time, the spacer maintains the probe in close proximity to the surface, ensuring high local concentration and effective target capture, thus preserving detection sensitivity while eliminating false positives
Solution Approach 2:
The probe structure is segmented into distinct functional domains including a target-binding domain, a spacer domain, and a reporter domain. The spacer domain specifically addresses the solid phase interaction issue by providing physical separation, while the target-binding domain maintains its ability to bind targets with high affinity. This segmentation allows independent optimization of each domain's function, enabling the probe to achieve both high sensitivity and high specificity on solid phase surfaces
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 method enhances the accuracy and convenience of detecting multiple target sequences on a solid phase by reducing false positives and enabling simultaneous detection of multiple sequences using a single label, improving reaction efficiency and optimization.
Implementation Method 1
cleaved by a 5' nuclease enzyme
Implementation Method 2
the released fragment is extended and hybridized with a CTO
Data Source
Figure 1A
Figure 1B
Figure 2A~2D
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a PCE-IH (PTO Cleavage and Extension-Dependent Immobilized Oligonucleotide Hybridization) assay on a solid phase. The present invention firstly hybridizes the PTO with a target nucleic acid sequence, forms the extended strand in a target-dependent manner by using the CTO having artificially selected sequence as templates and finally hybridizes the extended strand with the IO immobilized on a solid phase. In other words, the present invention employs a series of reactions including PTO hybridization and cleavage, CTO hybridization and extension and IO hybridization, which is responsible for the highly enhanced specificity of the present invention.