Isothermal Nucleic Acid Amplification via Recombinase Primer Segmentation
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Solution Overview
Problem
Current methods for nucleic acid amplification, such as PCR and LAMP, require temperature changes and multiple primers, which can be cumbersome and inefficient for generating multiple copies of nucleic acids for various applications.
Innovation Solution
A method involving the use of specific primers with defined regions for annealing and extension, allowing for the generation of concatemers by repeated primer extension and annealing steps without thermocycling, facilitating the amplification of nucleic acid templates at isothermal conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR method is used for nucleic acid amplification, then multiple copies of nucleic acid can be generated, but temperature changes are required which makes the process cumbersome and inefficient
Solution Approach 1:
The invention changes the temperature parameter from variable (PCR) to constant (isothermal), eliminating the need for thermal cycling while maintaining amplification capability through a different mechanism (recombinase-mediated strand invasion and primer extension at constant temperature)
Solution Approach 2:
The invention replaces the thermal cycling mechanism (mechanical heating/cooling system) with a biochemical mechanism (recombinase-primers system) that operates isothermally, substituting a complex mechanical temperature control system with a simpler biochemical reaction system
2Ease of operation
If LAMP method is used for nucleic acid amplification, then isothermal amplification is achieved, but four different primers are required which increases complexity
Solution Approach 1:
The invention segments the primer function into two distinct components: recombinase-primers (containing recombinase binding site and primer sequence) and extension primers (containing template binding site). This segmentation reduces the total number of primers needed while maintaining isothermal amplification capability
Solution Approach 2:
The recombinase-primers serve multiple functions: they bind to the recombinase binding site on the nucleic acid template, facilitate strand invasion through recombinase activity, and provide the 3' end for DNA polymerase extension. This multi-functionality reduces the number of separate reagents needed compared to LAMP
3Reliability
If multiple primers are used for amplification, then specific sequences can be recognized, but the number of primers increases which reduces efficiency
Solution Approach 1:
The recombinase-primers perform preliminary action by binding to the recombinase binding site and facilitating strand invasion before the extension primers bind to the template. This preliminary recognition step ensures sequence specificity is established early in the reaction, allowing subsequent amplification to proceed rapidly without requiring multiple different primers
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 efficiently generates multiple copies of nucleic acid templates, enhancing the amplification process by eliminating the need for temperature changes and reducing the complexity of primer usage, thereby improving the yield and simplicity of nucleic acid amplification.
Implementation Method 1
treating a primary double-stranded nucleic acid comprising the double-stranded nucleic acid template with a first copy of a first primer and a polymerase under conditions such that an extension product of the first copy of the first primer is synthesized
Implementation Method 2
the template-binding region of the first copy of the first primer anneals to the first strand of the double-stranded nucleic acid template
Data Source
AI summary
Methods and compositions for the amplification of nucleic acids and generation of concatemers are disclosed. Amplification methods provided herein may be performed under isothermal conditions. Methods and compositions may include reagents such as nucleic acid polymerases and primers.