PCEC Nucleic Acid Detection via Sequential Cleavage
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Solution Overview
Problem
Conventional DNA hybridization-based methods for detecting target nucleic acid sequences are prone to false positive results due to non-specific hybridization and are limited by the need for specific reaction conditions and types of labels, particularly fluorescent labels, which complicates the detection process.
Innovation Solution
The PCEC (PTO Cleavage and Extension-Dependent Cleavage) assay, which involves hybridizing a target nucleic acid sequence with a PTO and CTO oligonucleotides, followed by successive cleavage reactions using 5' nuclease activity and nucleolytic enzymes, allowing for detection in both liquid and solid phases with improved accuracy and convenience, and the ability to detect multiple sequences without being limited by the type of labels used.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional DNA hybridization-based methods are used for detection, then the detection process is simple, but false positive results occur due to non-specific hybridization
Solution Approach 1:
The detection process is divided into multiple sequential steps: initial hybridization, first cleavage reaction, second hybridization, and second cleavage reaction. Each step builds upon the previous one, creating a cascaded detection system where the target sequence must satisfy multiple conditions to generate a signal, thereby reducing false positives while maintaining procedural clarity
Solution Approach 2:
The first cleavage reaction acts as a preliminary filtering step before the second hybridization and cleavage. By requiring the target sequence to successfully complete the first cleavage event, the system pre-validates the specificity of binding before generating the final detection signal, reducing false positives without requiring complex real-time monitoring
2Reliability
If TaqMan probe method with polymerization-dependent cleavage is used, then signal generation is achieved, but the upstream primer must be extended before the polymerase can cleave the probe
Solution Approach 1:
The detection assay is segmented into distinct cleavage events separated by hybridization steps. The first cleavage reaction occurs independently of primer extension, and the second cleavage reaction occurs after a separate hybridization event. This segmentation eliminates the requirement for the polymerase to extend the upstream primer before cleaving the probe, as cleavage is triggered by hybridization rather than extension
Solution Approach 2:
Instead of requiring primer extension to bring the polymerase into position for probe cleavage (conventional TaqMan approach), the invention inverts the mechanism by using hybridization of the cleaved fragment to a second probe to position the system for the second cleavage event. This reverses the causal sequence from extension-then-cleavage to hybridization-then-cleavage
3Adaptability or versatility
If multiple target sequences are detected using conventional methods, then detection capability is expanded, but the type of labels used is limited particularly fluorescent labels
Solution Approach 1:
The assay design uses universal cleavage mechanisms that can be applied to different probe sequences without requiring different label types. The same enzymatic cleavage chemistry and detection principles work across multiple target sequences, allowing multiplex detection to be achieved by varying probe sequences rather than being constrained to specific label types, thereby expanding versatility while maintaining 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
This method enhances the accuracy and convenience of target sequence detection, enabling reliable and reproducible multiplex detection of nucleic acid sequences by generating a target signal through successive cleavage reactions, thereby reducing false positives and expanding label flexibility.
Implementation Method 1
contacting the resultant of the 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
performing an extension reaction using the resultant of the 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 3
cleaving the extended duplex using the nucleolytic enzyme to form a cleaved fragment; wherein the occurrence of the cleavage of the extended duplex indicates the presence of the target nucleic acid sequence
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a PCEC (PTO Cleavage and Extension-Dependent Cleavage) assay. The present invention is characterized by generating a cleavage site for a nucleolytic enzyme on the extended duplex of which the formation is dependent on the presence of a target nucleic acid sequence. The present invention detects the occurrence of the cleavage of the extended duplex, thereby determining the presence of the target nucleic acid sequence.