PTO Cleavage and Extension Assay for Specific Multiplex DNA Detection
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Solution Overview
Problem
Conventional nucleic acid sequence detection methods, such as hybridization and TaqMan™ probe methods, suffer from false positives due to non-specific hybridization and require multiple fluorescent labels for multiplex target detection, limiting the number of sequences that can be detected and complicating solid-phase reactions.
Innovation Solution
The PTOCE (PTO Cleavage and Extension) assay combines probe hybridization with enzymatic cleavage and extension reactions to detect target nucleic acid sequences, allowing for multiplex detection in both liquid and solid phases without the need for multiple fluorescent labels, using PTO and CTO oligonucleotides with specific structural features and enzymatic cleavage by 5' nuclease activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If TaqMan probe method is used for nucleic acid detection, then detection sensitivity is improved, but false positive results occur due to non-specific hybridization
Solution Approach 1:
The probe is divided into three functional segments: a 5' tail region (non-complementary to target), a middle region (complementary to target for hybridization), and a 3' region (for polymerase binding). This segmentation allows the probe to undergo conformational change from open to closed state upon target binding, enabling specific cleavage only when all regions are properly positioned, thus reducing false positives while maintaining sensitivity
Solution Approach 2:
The probe is pre-designed with a 5' tail region that is non-complementary to the target sequence. This preliminary structural arrangement ensures that the probe can only be cleaved by the polymerase's 5' exonuclease activity after it has first hybridized to the target through the middle region and positioned the 3' region for polymerase binding, providing an additional layer of specificity before the detection signal is generated
2Adaptability or versatility
If multiple fluorescent labels are used for multiplex detection, then the number of detectable sequences increases, but device complexity and cost increase
Solution Approach 1:
The invention uses a universal detection mechanism based on fluorescence resonance energy transfer (FRET) between a donor fluorophore and various acceptor molecules. The same donor fluorophore can work with different acceptors (quencher, fluorophore, or absorbent) to detect different target sequences, allowing multiplex detection without requiring multiple different donor fluorophores, thus reducing complexity while maintaining versatility
Solution Approach 2:
The invention introduces an intermediary detection mechanism using FRET pairs where the donor fluorophore on the probe transfers energy to an acceptor molecule whose identity determines the detection channel. This intermediary energy transfer mechanism allows the same probe structure to be used across multiple detection channels by simply changing the acceptor, reducing the need for multiple complex labeled probes
3Ease of operation
If conventional hybridization methods are used, then detection process is simple, but specificity is reduced due to non-specific hybridization
Solution Approach 1:
The invention replaces the passive hybridization-based detection mechanism with an active enzymatic cleavage mechanism. Instead of relying solely on the stability of the hybridized probe-target complex, the system uses the polymerase's 5' exonuclease activity to cleave the probe only when it is properly hybridized to the target, providing an active verification step that enhances specificity while maintaining operational simplicity
Solution Approach 2:
The invention changes the detection parameter from measuring hybridization stability (melting temperature) to measuring enzymatic cleavage activity. By monitoring the fluorescence signal generated from probe cleavage rather than hybridization melting, the system achieves higher specificity because cleavage only occurs when the probe is correctly positioned on the target through proper hybridization of all three regions
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 PTOCE assay provides accurate and convenient detection of multiple target sequences by forming an extended duplex with adjustable Tm values, enabling reliable discrimination and detection through melting analysis or temperature-dependent detection.
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 and an extended duplex is formed
Implementation Method 4
melting the extended duplex over a range of temperatures to give a target signal indicative of the presence of the extended duplex
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a PTOCE (PTO Cleavage and Extension) assay. The present invention detects a target nucleic acid sequence in which the PTO (Probing and Tagging Oligonucleotide) hybridized with the target nucleic acid sequence is cleaved to release a fragment and the fragment is hybridized with the CTO (Capturing and Templating Oligonucleotide) to form an extended duplex, followed by detecting the presence of the extended duplex. The extended duplex provides signals (generation, increase, extinguishment or decrease of signals) from labels indicating the presence of the extended duplex and has adjustable Tm value, which are well adoptable for detection of the presence of the target nucleic acid sequence.