PCE-hCTO Assay Solid Phase Nucleic Acid Detection
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Solution Overview
Problem
Conventional DNA hybridization-based methods for detecting target nucleic acid sequences on a solid phase often produce false positive results due to non-specific hybridization and require multiple fluorescent labels for multiplex target detection, limiting their accuracy and efficiency.
Innovation Solution
The PCE-hCTO (PTO Cleavage and Extension using hCTO) assay involves probe hybridization and enzymatic reactions, including 5' nucleolytic reactions, to form an extended duplex on a solid phase, allowing for the detection of multiple target sequences with improved accuracy and convenience using a single label.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional DNA hybridization-based methods are used for detection on solid phase, 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, a cleavage site, and a reporter domain. This segmentation allows the probe to perform multiple functions (hybridization, cleavage, signal generation) in sequence, improving reliability by requiring multiple specific interactions rather than relying solely on hybridization specificity
Solution Approach 2:
The probe is pre-designed with a cleavage site and reporter moiety attached to the 5'-tail region. When the probe hybridizes to the target sequence, the cleavage is immediately triggered, releasing the reporter. This preliminary arrangement of components ensures that signal generation is coupled directly to specific hybridization events, reducing false positives
2Adaptability or versatility
If multiple fluorescent labels are used for multiplex target detection, then multiple target sequences can be detected, but the cost and complexity increase
Solution Approach 1:
A single fluorescent label attached to the 5'-tail region serves multiple purposes: it provides the detection signal and enables differentiation between multiple target sequences through variations in melting temperature (Tm). The same label can be used across different probe designs, and multiplexing is achieved by adjusting probe sequences and lengths rather than using different labels
Solution Approach 2:
Instead of changing the fluorescent label to detect different targets, the invention changes physical parameters of the probe (sequence composition, length, GC content) to create different Tm values. This allows a single label to differentiate between multiple targets based on their unique thermal denaturation characteristics, simplifying the labeling system while maintaining multiplex capability
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 overcoming limitations of non-specific hybridization and label limitations, enabling adjustable Tm values for effective signal differentiation and detection.
Implementation Method 1
DNA hybridization is a fundamental process in molecular biology and is affected by ionic strength, base composition, length of fragment to which the nucleic acid has been reduced, the degree of mismatching, and the presence of denaturing agents
Implementation Method 2
the labeled probe hybridized with a target nucleic acid sequence is cleaved by a 5′ nuclease activity of an upstream primer-dependent DNA polymerase, generating a signal indicating the presence of a target sequence
Implementation Method 3
In TaqMan probe method, the labeled probe hybridized with a target nucleic acid sequence is cleaved by a 5′ nuclease activity of an upstream primer-dependent DNA polymerase
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence from a DNA or a mixture of nucleic acids by a PCE-hCTO (PTO Cleavage and Extension using hCTO) assay on a solid phase. According to the present invention, the extended duplex is formed in a liquid phase in a target-dependent manner and then its presence is detected on a solid phase. Since hCTO is not immobilized onto a solid phase, the extended duplex is more effectively formed in a liquid phase.