Q-PTOCE Assay for Single-Temperature Multiplex Nucleic Acid Detection
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Solution Overview
Problem
Conventional real-time nucleic acid detection methods are limited in their ability to simultaneously detect multiple target nucleic acids due to the restriction of the number of labels used, and melting analysis becomes cumbersome with increasing targets.
Innovation Solution
A novel Q-PTOCE assay utilizing a primer, Quenching-Probing and Tagging Oligonucleotide (Q-PTO) and Capturing and Templating Oligonucleotide (CTO) with quencher and reporter molecules, enabling detection through hybridization, cleavage, and extension reactions to generate an extended strand indicative of target nucleic acid presence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional real-time detection methods using labeled probes or primers are used, then detection of target nucleic acid can be achieved, but the number of target nucleic acids that can be simultaneously detected is limited by the number of labels (5 or less)
Solution Approach 1:
The patent applies universality by designing a single-label system that can detect multiple target nucleic acids simultaneously. The Q-PTO and CTO oligonucleotides serve multiple functions: they enable specific binding to different targets, provide signal generation through cleavage, and allow temperature-dependent signal discrimination. This multi-functional design eliminates the need for multiple labels while maintaining the ability to detect multiple targets.
Solution Approach 2:
The patent utilizes parameter changes by detecting signals at different temperatures. Each target nucleic acid produces a signal at a specific detection temperature based on the Tm of the corresponding extended duplex. By varying the detection temperature parameter, the system can selectively detect different targets using the same label, thereby increasing the quantity of detectable targets without proportionally increasing device complexity.
2Quantity of substance
If melting analysis is used to detect multiple target nucleic acids using a single label, then the number of detectable targets increases, but the detection time increases significantly and probe design becomes increasingly difficult
Solution Approach 1:
The patent implements continuous useful action by performing detection in real-time during the amplification process. The Q-PTOCE assay allows for continuous monitoring of target amplification and simultaneous detection without requiring separate melting analysis steps. The signal generation occurs continuously as the DNA polymerase extends the primer and cleaves the Q-PTO, eliminating the need for time-consuming post-amplification melting analysis.
3Quantity of substance
If melting analysis is used to detect multiple target nucleic acids, then detection capability increases, but probe design with different Tm values becomes increasingly difficult
Solution Approach 1:
The patent applies segmentation by dividing the detection system into modular components: the Q-PTO with its 5'-tagging portion and 3'-targeting portion, and the CTO with its capturing portion and templating portion. Each component can be independently designed and optimized. The tagging portions can be standardized while the targeting portions are customized for different targets, simplifying the overall design process compared to creating entirely different probes for each target with specific Tm values.
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
Enables simultaneous detection of multiple nucleic acids at a single temperature in a single reaction vessel by adjusting the signal detection temperature through duplex dissociation, overcoming limitations of conventional methods.
Implementation Method 1
the reporter molecule is in close proximity to the first quencher molecule, thereby causing the reporter molecule to be quenched by the first quencher molecule
Implementation Method 2
contacting the resultant of the step (a) to a DNA polymerase having 5' nuclease activity under conditions for cleavage of the Q-PTO
Implementation Method 3
hybridizing a primer and a Quenching-Probing and Tagging Oligonucleotide (Q-PTO) with the target nucleic acid
Data Source
Figure 1(a)~1(e)
Figure 2(a)~2(b)
Figure 3(a)~3(b)
AI summary
The present disclosure relates to a method for detecting a target nucleic acid in a sample by Quenching-PTO Cleavage and Extension (Q-PTOCE) assay. The present disclosure has an advantage in that the signal detection temperature can be selected within a higher temperature range by utilizing not only the cleavage of the Q-PTO and hybridization between the reporter-carrying fragment and the CTO, but also the extension of the reporter-carrying fragment after hybridization