PTO Cleavage and Extension-Dependent Non-Hybridization Assay
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Solution Overview
Problem
Conventional DNA hybridization-based methods for detecting target nucleic acid sequences are prone to false positives due to non-specific hybridization and require multiple fluorescent labels for multiplex target detection, limiting their reliability and efficiency.
Innovation Solution
The PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay involves probe hybridization, enzymatic probe cleavage, and extension using a hybridizing oligonucleotide (HO), allowing for the detection of multiple target sequences in both liquid and solid phases with improved accuracy and convenience, without the need for multiple fluorescent labels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If DNA hybridization-based methods are used for target sequence detection, then the detection process is simple, 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 hybridizes to the target sequence, and a signal-generation domain that remains single-stranded. This segmentation allows the probe to distinguish between specific and non-specific binding, eliminating false positives while maintaining operational simplicity
Solution Approach 2:
A blocking oligonucleotide is introduced as an intermediary element that competes with non-target sequences for probe binding. This intermediary prevents non-specific hybridization by occupying the probe's binding site when the target is absent, thereby improving detection reliability without complicating the overall process
2Adaptability or versatility
If multiple fluorescent labels are used for multiplex target detection, then multiple targets can be detected simultaneously, but the device complexity and cost increase
Solution Approach 1:
A single fluorescent label is designed to serve multiple functions across different detection scenarios. The probe structure enables discrimination between one target and multiple targets using the same label, allowing multiplex detection without requiring multiple different fluorescent markers, thus reducing device complexity while maintaining versatility
Solution Approach 2:
The detection system utilizes changes in physical parameters (such as fluorescence intensity, melting temperature, or hybridization kinetics) rather than relying on multiple fluorescent labels. By monitoring parameter changes in response to different target-probe interactions, the system achieves multiplex detection capability with a single label, simplifying the overall detection system
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 target sequence detection by preventing hybridization between the CTO and HO only when the target sequence is present, using a single label or no label at all, thereby overcoming the limitations of conventional methods.
Implementation Method 1
contacting the resultant of step (a) to an enzyme having a 5′ nuclease activity under conditions for cleavage of the PTO; wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the enzyme having the 5′ nuclease activity
Implementation Method 2
hybridizing the fragment released from the PTO with a CTO (Capturing and Templating Oligonucleotide)
Implementation Method 3
performing an extension reaction using the resultant of step (c) and a template-dependent nucleic acid polymerase; wherein the fragment hybridized with the capturing portion of the CTO is extended to form an extended duplex
Implementation Method 4
when the target nucleic acid sequence is present in the nucleic acid sample, the extended duplex is formed to prevent the formation of the hybrid between the CTO and the HO
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a PCE-NH (PTO Cleavage and Extension-Dependent Non-Hybridization) assay. The present invention adopts the occurrence of the inhibition of the hybridization between the HO with the CTO by the formation of the target-dependent extended duplex. Therefore, the present invention may detect target sequences even when the HO is not cleaved. In this regard, the design of the 5′-tagging portion of PTO, CTO and HO sequences may be readily performed and the conditions for reactions may be also easily established. In addition, the detection of the hybrid between the CTO and the HO may be performed in a different vessel from that for the extension of the CTO.