Resolvase Cleavage of Primer-Dimers in Multiplex PCR
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Solution Overview
Problem
Current multiplex PCR methods face challenges in effectively reducing non-specific amplification products, such as primer-dimers, which overwhelm target-specific amplification products, limiting the number of targets that can be simultaneously amplified and hindering diagnostic applications.
Innovation Solution
The use of resolvases like T4 endonuclease VII and T7 endonuclease I to specifically cleave aberrant DNA structures in non-specific amplification products, allowing for the preservation of target-specific amplification products during multiplex PCR reactions, even with high numbers of primer pairs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a large number of primer pairs are used in multiplex PCR to amplify multiple target fragments simultaneously, then the throughput and productivity are improved, but the formation of non-specific amplification products (primer-dimers) increases and overwhelms the target-specific products
Solution Approach 1:
The patent extracts and removes non-specific amplification products from the reaction mixture using specific enzymes (exonucleases, nucleases, or resolvases) that selectively degrade primer-dimers and non-specific products while leaving target-specific amplification products intact. This allows the system to maintain high primer concentrations for high throughput while eliminating the harmful background noise they generate
Solution Approach 2:
The patent introduces intermediary enzymes (exonucleases, nucleases, or resolvases) that act as mediators between the primer pairs and the amplification products. These enzymes selectively recognize and degrade non-specific products formed by primer interactions, thereby enabling the use of high primer concentrations without being overwhelmed by non-specific amplification
2Productivity
If primer concentrations are increased to amplify more targets in a single reaction, then the productivity and efficiency are improved, but the primers accumulate to very high quantities that lead to extensive primer-annealing and non-specific product formation
Solution Approach 1:
The patent converts the harmful effect of high primer concentrations (which cause non-specific annealing) into a beneficial outcome by using the resulting non-specific products as substrates for selective degradation enzymes. The enzymes recognize and degrade these non-specific products, thereby allowing the system to tolerate and even utilize high primer concentrations for high-throughput amplification without being overwhelmed by background noise
3Object-generated harmful factors
If current techniques such as specific primer design or use of oligonucleotides to prevent primer-dimer formation are employed, then the non-specific product formation is reduced, but the device complexity and difficulty of primer design increase substantially
Solution Approach 1:
The patent employs a self-service approach where the system automatically handles non-specific product formation and removal without requiring complex pre-design of primers. Standard primer design protocols can be used, and the system self-corrects by using enzymes to selectively degrade any non-specific products that form during amplification, thereby simplifying the overall process while maintaining effectiveness
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 significantly reduces non-specific amplification products while maintaining a substantial proportion of target-specific products, enabling the simultaneous amplification of thousands of target-specific nucleic acid molecules in a single reaction, enhancing the reliability and efficiency of multiplex PCR.
Implementation Method 1
The use of resolvases like T4 endonuclease VII and T7 endonuclease I to specifically cleave aberrant DNA structures in non-specific amplification products
Data Source
AI summary
Methods, compositions, systems and kits to amplify or improve amplification of target-specific amplification products by reducing non-specific amplification products (e.g., primer-dimers) when amplifying multiple different nucleotide regions. The methods, compositions, systems and kits described herein may include, or include the use of, one or more resolvases that recognize and bind to and/or cut an aberrant DNA structure.