SCORPION Primer Stem-Loop Extraction for Polymorphic Detection
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Solution Overview
Problem
Existing nucleic acid detection methods face challenges in designing primers for highly polymorphic organisms due to the difficulty in finding conserved sequences, especially for those requiring three unique conserved sequences, which limits flexibility and accuracy in amplification reactions.
Innovation Solution
The development of nucleic acid-based detection mechanisms involving a first and second nucleic acid sequence connected by a non-nucleic acid linker with a signaling element, allowing for hybridization and signal changes when not hybridized, enabling detection and amplification of specific sequences with improved flexibility and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If SCORPION primers with stem-loop structure are used, then specific binding to amplified product is achieved, but requirement for three unique conserved sequences makes design difficult in highly polymorphic organisms
Solution Approach 1:
The patent extracts the stem-loop structure from the primer design, separating the detection function (stem-loop) from the binding function (primer). This allows the primer to bind to a single conserved sequence while the stem-loop provides detection capability, eliminating the need to find three conserved sequences while maintaining specific binding through the extracted stem-loop element.
Solution Approach 2:
The detection mechanism is segmented into separate functional components: the primer portion for binding to the target sequence and the stem-loop detection portion. This segmentation allows independent optimization of binding specificity (through primer design) and detection reliability (through stem-loop structure), resolving the contradiction between reliable detection and ease of design.
2Device complexity
If AMPLIFLUOR primers with hairpin structure are used, then detection mechanism is simplified, but requires conserved sequence sufficiently long to bind both primer and stem-loop structure reducing flexibility
Solution Approach 1:
The patent extracts the stem-loop structure from the primer sequence itself, placing it as a separate detection component. This allows the primer to be designed based on minimal conserved sequences (improving flexibility) while the separate stem-loop provides the necessary detection mechanism (maintaining simplicity).
Solution Approach 2:
The patent introduces a non-nucleic acid linker as an intermediary between the primer and the stem-loop structure. This linker allows the two components to function independently - the primer binds to short conserved sequences while the stem-loop provides detection - thereby maintaining design flexibility without compromising detection mechanism simplicity.
3Adaptability or versatility
If primers targeting short conserved regions are used, then flexibility in highly polymorphic organisms is improved, but detection sensitivity may be reduced
Solution Approach 1:
The non-nucleic acid linker acts as an intermediary that decouples the binding region (primer) from the detection region (stem-loop). This allows the primer to target short conserved regions for flexibility while the stem-loop, positioned via the linker, provides enhanced detection sensitivity through its structured configuration, resolving the contradiction between flexibility and sensitivity.
Solution Approach 2:
The patent applies local quality by concentrating different functional properties in different locations: the primer region provides binding specificity to short conserved sequences, while the stem-loop region provides detection sensitivity. The non-nucleic acid linker positions these functional elements optimally, allowing each to excel at its specific function without compromising the other.
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 enhances the ability to detect and quantify nucleic acid amplification products, particularly in highly polymorphic organisms, by targeting short conserved regions and reducing false negatives, applicable to various nucleic acid amplification technologies.
Implementation Method 1
a second nucleic acid sequence sufficiently complementary to at least a portion of the first nucleic acid sequence to allow formation of a double-stranded DNA molecule under normal DNA hybridization conditions
Implementation Method 2
The signaling element may include, for example, a single signaling molecule, a pair of signaling molecules, a signaling molecule and a quenching molecule or other signaling mechanisms. A signal from the signaling element may detectably change when the first and second nucleic acid sequences are not hybridized to one another.
Data Source
AI summary
Disclosed are methods and compositions related to real-time PCR and other nucleic acid extension or amplification reactions.