PTOCE-E Nucleic Acid Detection Reducing Non-Target Signals
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Solution Overview
Problem
Existing nucleic acid detection methods face challenges in reducing non-target signals while maintaining target signals, particularly in multiplex amplification reactions, leading to decreased accuracy and efficiency.
Innovation Solution
The PTOCE-E assay method involves probe hybridization, primary enzymatic cleavage, and secondary enzymatic extension reactions, using specific oligonucleotides to detect target nucleic acid sequences through successive hybridization and extension steps, enhancing accuracy and convenience in both liquid and solid phase reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional real-time detection methods using labeled probes or primers are used, then target nucleic acid sequences can be detected, but non-target signals increase particularly in multiplex amplification reactions
Solution Approach 1:
The detection system is divided into multiple functional components: a first probe with 5'-reporter and 3'-quencher labels, a second probe with internal labels, and a third probe for multiplex detection. Each probe segment performs a specific function (signal generation, quenching, or target recognition), allowing precise control over signal generation and reducing non-specific signals through functional division of labor
Solution Approach 2:
Different regions of the probe molecules are assigned different properties: the 5'-end contains reporter labels for signal generation, the 3'-end contains quencher labels for signal suppression, and internal regions contain sequence-specific binding domains. This local differentiation allows the probe to simultaneously generate and control signals, improving detection accuracy while minimizing non-target signals
2Adaptability or versatility
If multiple target sequences are detected in multiplex reactions, then detection versatility improves, but non-target signals increase and accuracy decreases
Solution Approach 1:
The probe system is designed with universal functional elements that can detect multiple target sequences: the first probe structure with 5'-reporter and 3'-quencher can bind to different target sequences by changing only the central binding region, while maintaining the same signal generation mechanism. This allows a single probe design framework to universally detect multiple targets with equal accuracy
Solution Approach 2:
The second probe acts as an intermediary that specifically binds to the first probe only when both are hybridized to their respective target sequences. This intermediary mechanism ensures that signal amplification occurs only when specific target sequences are present, preventing false signals in multiplex reactions while maintaining high detection versatility
3Measurement precision
If probe hybridization and enzymatic reactions are used for detection, then detection sensitivity improves, but reaction complexity and time increase
Solution Approach 1:
The detection method merges probe hybridization, enzymatic cleavage, and fluorescent signal generation into a single integrated reaction system. The first probe, second probe, and third probe work together in one reaction mixture with a single enzymatic step, eliminating the need for separate preparation and analysis steps, thus maintaining high sensitivity while reducing overall process complexity
Solution Approach 2:
The probe system is designed to be self-sufficient: the probes contain all necessary functional elements (reporter labels, quencher labels, binding sequences) within their structures, and the enzymatic reaction automatically generates the detection signal without requiring additional reagents or separate processing steps. This self-service design simplifies the overall detection process while maintaining high sensitivity
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 approach improves the detection of target sequences by reducing non-target signals and increasing accuracy, allowing for the reliable identification of multiple target sequences in multiplex reactions.
Implementation Method 1
contacting the resultant of step (a) to a 5'-nuclease enzyme under conditions for cleavage of the PTO; wherein the upstream oligonucleotide or its extended strand induces cleavage of the PTO by the 5'-nuclease enzyme
Implementation Method 2
performing an extension reaction using the resultant of step (c) and a template-dependent nucleic acid polymerase; wherein the PTO fragment hybridized with the capturing portion of the first CTO is extended to generate a first extended strand comprising a first extended sequence complementary to the templating portion of the first CTO
Implementation Method 3
hybridizing the first extended strand with a second CTO; wherein the second CTO comprises in a 3' to 5' direction (i) a capturing portion comprising a hybridizing nucleotide sequence to the first extended strand and (ii) a templating portion comprising a non-hybridizing nucleotide sequence to the first extended strand
Data Source
AI summary
The present invention relates to the detection of a target nucleic acid sequence by a PTOCE-E (PTO Cleavage and Extension-dependent Extension) assay. The PTOCE-E assay of the present invention can reduce the non-target signal and increase the target signal as compared with the conventional PTOCE and PCE-SH methods, thereby enabling more accurate detection of the target nucleic acid sequence.