PO Cleavage and Hybridization Assay for Nucleic Acid Detection
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional DNA hybridization-based technologies on solid substrates face challenges in efficiently detecting target nucleic acid sequences due to limitations in probe design and reaction conditions, particularly when using tag sequence-carrying probes and capture probes immobilized on solid substrates, leading to reduced accuracy and convenience.
Innovation Solution
The development of a novel POCH (PO Cleavage and Hybridization) assay using probing oligonucleotides (PO) and capturing oligonucleotides (CO), where target detection is achieved through a probe-cleavage reaction and additional probe-hybridization, allowing for enhanced specificity and accuracy on solid phases without the need for complex probe design or orientation control.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If tag sequence-carrying probes and capture probes are used on solid substrates, then target detection capability is provided, but probe design complexity and orientation control requirements increase
Solution Approach 1:
The invention extracts the tag sequence from the probe structure itself and places it on the solid substrate as a separate capture probe. This separates the detection function (probe hybridization) from the capture function (tag binding), eliminating the need for complex probe design with integrated tags and reducing orientation control requirements.
2Reliability
If conventional probe hybridization methods are used on solid substrates, then target detection is achieved, but accuracy is reduced due to limitations in probe design and reaction conditions
Solution Approach 1:
The invention segments the detection process into two independent steps: (1) probe hybridization with target sequence under optimized conditions, and (2) capture probe binding to tag sequences on the solid substrate. This segmentation allows each step to be optimized independently, improving overall accuracy while simplifying reaction condition control.
Solution Approach 2:
The tag sequence acts as an intermediary between the probe-target hybrid and the solid substrate. Instead of directly controlling probe orientation on the substrate, the tag-mediated capture probe provides a simple binding interface that enhances accuracy without complicating reaction conditions.
3Adaptability or versatility
If multiple target sequences need to be detected, then detection versatility is required, but conventional methods become less convenient and more complex
Solution Approach 1:
The invention creates a universal detection platform where the solid substrate contains capture probes for multiple different tag sequences. A single probe hybridization step can detect multiple target sequences simultaneously by binding to different tags on the substrate, enabling multiplex detection while maintaining operational simplicity.
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
The POCH assay provides improved accuracy and convenience in detecting target sequences, enabling multiple detections with enhanced specificity and simplifying reaction conditions, overcoming the limitations of conventional methods.
Implementation Method 1
the upstream oligonucleotide or its extended strand induces cleavage of the PO by an enzyme having a 5' nuclease activity
Implementation Method 2
performing a hybridization reaction by contacting the resultant of the step (b) to a capturing oligonucleotide (CO) immobilized onto the solid substrate
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a POCH (PO Cleavage and Hybridization) assay on a solid substrate. The present invention detects the target nucleic acid sequence by use of in which the PO (Probing Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved and the cleavage of the PO is detected by hybridization with the CO (Capturing Oligonucleotide). In the present invention, an uncleaved PO is hybridized with the CO immobilized onto the solid substrate. The designs of the PO and the CO are convenient and the optimization of reaction conditions is routinely easy in the present invention. Where the detection of signal on the solid substrate is continuously performed along with repetition of cleavage of the POs in the present invention, the number of the POs cleaved is increased upon the repetition number of the cleavage reaction and the signal is changed in parallel with the number of the POs cleaved. Then, the target nucleic acid sequence can be detected in a real-time manner. In contrast, the change of the signal is not observed in the absence of the target nucleic acid sequence.