Tag-Mediated Displacement Nucleic Acid Amplification
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Solution Overview
Problem
Current nucleic acid amplification methods, such as PCR, require temperature cycling and may not be efficient for detecting and quantitating specific microorganisms in various samples, particularly in isothermal conditions.
Innovation Solution
The method employs amplification oligonucleotides with heterologous displacer tags and universal tags to initiate and sustain nucleic acid amplification reactions, allowing for geometric amplification without the need for temperature cycling, using a sequence-based mechanism that incorporates displacer and universal priming segments to enhance amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If temperature cycling is used for nucleic acid amplification (PCR), then amplification can be achieved, but the process becomes complex and less efficient for isothermal conditions
Solution Approach 1:
The patent replaces the mechanical temperature cycling system with a chemical displacement mechanism. Instead of using thermal cycles to drive amplification, the invention uses a chemical displacement reaction where a displacer oligonucleotide replaces the extended primer strand, enabling amplification under constant temperature conditions and eliminating the need for complex temperature cycling equipment.
Solution Approach 2:
The patent changes the fundamental parameter driving amplification from temperature to chemical displacement. By introducing a displacer tag and displacer oligonucleotide that mediate strand displacement, the system transitions from thermal-driven PCR to chemically-driven amplification, allowing efficient amplification at constant temperatures without requiring temperature cycling.
2Measurement precision
If traditional PCR methods are used, then nucleic acid amplification can be performed, but sensitivity and specificity for detecting microorganisms in various samples is limited
Solution Approach 1:
The patent introduces a displacer tag as an intermediary element that enhances amplification reliability. The displacer tag on the amplification oligonucleotide mediates the displacement reaction, ensuring efficient and specific amplification. This intermediary mechanism improves both sensitivity and reliability by facilitating more accurate and consistent target nucleic acid amplification compared to traditional PCR.
Solution Approach 2:
The patent segments the amplification oligonucleotide into distinct functional regions: a target-binding segment for specific binding to the microorganism's nucleic acid, and a displacer tag segment that mediates the displacement reaction. This segmentation allows each region to optimize its specific function, improving overall detection sensitivity and amplification reliability for microorganism identification.
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 enables robust and sensitive nucleic acid amplification under constant temperature conditions, overcoming the limitations of traditional methods by achieving efficient amplification of target nucleic acid regions with improved specificity and sensitivity.
Implementation Method 1
The method employs amplification oligonucleotides with heterologous displacer tags and universal tags to initiate and sustain nucleic acid amplification reactions, allowing for geometric amplification without the need for temperature cycling, using a sequence-based mechanism that incorporates displacer and universal priming segments to enhance amplification efficiency.
Implementation Method 2
primer extension in the presence of a dNTP to produce a duplex hemiphosphorothioated primer extension product
Data Source
AI summary
Disclosed are methods for amplifying a nucleic acid target region using an amplification oligomer comprising a target-binding segment and a heterologous displacer tag situated 5′ to the target-binding segment. Initiation of an amplification reaction from the tagged amplification oligomer produces an amplicon comprising the displacer tag, such that once the complement of the displacer tag has been incorporated into a second amplicon, a displacer oligonucleotide having a sequence substantially corresponding to the displacer tag sequence is used to participate in subsequent rounds of amplification for displacement of an extension product primed from a site within the second amplicon 5′ to the displacer priming site. Also disclosed are related kits and reaction mixtures comprising the displacer-tagged amplification oligomer and corresponding displacer oligonucleotide.