PTOCE Assay for Nucleotide Variation Detection
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Solution Overview
Problem
Conventional DNA hybridization-based methods for detecting nucleotide variations are prone to false positives due to non-specific hybridization and require multiple fluorescent labels, limiting their reliability and efficiency in multiplex target detection.
Innovation Solution
The PTOCE (Probing and Tagging Oligonucleotide Cleavage and Extension) assay, which involves hybridizing a target nucleic acid sequence with a PTO and CTO, using enzymatic cleavage and extension reactions to detect nucleotide variations, allowing for adjustable cleavage sites and label-independent detection of multiple targets in both liquid and solid phases.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
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 probe is divided into multiple functional segments: a 5' non-complementary region, a 3' complementary region, and an internal cleavage site. This segmentation allows the probe to undergo specific cleavage only when bound to the target, enabling discrimination between specific and non-specific hybridization events.
Solution Approach 2:
A cleavage enzyme serves as an intermediary that mediates between probe hybridization and signal generation. The enzyme specifically cleaves the probe at the internal site only when the probe is correctly hybridized to the target, providing an additional specificity layer that eliminates false positives from non-specific hybridization.
2Adaptability or versatility
If multiple fluorescent labels are used for multiplex detection, then multiple targets can be detected simultaneously, but the complexity and cost increase
Solution Approach 1:
The invention uses universal unlabeled probes combined with a universal cleavage enzyme that can detect multiple different targets. The specificity is achieved through the hybridization complementarity rather than fluorescent labels, allowing one probe design to work across multiple targets without requiring different labeled probes for each target.
Solution Approach 2:
The invention replaces the optical detection system (multiple fluorescent labels and filters) with a biochemical system (cleavage enzyme activity). Instead of detecting multiple fluorescent signals, the system uses a single cleavage reaction that can be detected by various methods, simplifying the detection apparatus while maintaining multiplex capability.
3Measurement precision
If TaqMan probe method is used, then signal generation is enhanced, but the requirement for upstream primer-dependent polymerase limits applicability
Solution Approach 1:
The invention extracts and utilizes the 5' to 3' exonuclease activity of DNA polymerase independently from the primer extension function. By designing the probe with a 5' non-complementary region and internal cleavage site, the probe can be cleaved by the exonuclease activity without requiring upstream primer extension, thereby separating these two functions and broadening method applicability.
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 nucleotide variation detection, enabling reliable and reproducible multiplex analysis without the need for multiple fluorescent labels, and allows for adjustable Tm values to discriminate between target and non-target signals.
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
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 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
Data Source
Figure 1A~1B
Figure 2A~2E
Figure 3A~3E
AI summary
The present invention is generally drawn to a novel method and a kit for detecting nucleotide variations by a PTOCE (PTO Cleavage and Extension) assay with PTO-NV. Furthermore, the present invention is directed to a novel method and a kit for detecting a nucleotide variation on a target nucleic acid sequence by a PTOCE assay with PTO-NV having a non-base paring moiety.