Isothermal Nucleic Acid Amplification via Primer Segmentation
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Solution Overview
Problem
Current nucleic acid amplification methods, such as PCR and LAMP, require temperature changes and multiple primers, which can be cumbersome and inefficient, highlighting the need for new methods and reagents that facilitate the amplification of nucleic acids for various applications.
Innovation Solution
A method involving the use of specific primers with defined regions and a polymerase to generate concatemers by repeated extension and annealing processes, allowing for the amplification of nucleic acid templates at controlled temperatures without the need for temperature cycling, using primers with 5' and 3' terminal nucleotides and specific tail and template-binding regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR method is used for nucleic acid amplification, then amplification efficiency is improved, but temperature cycling hardware complexity increases
Solution Approach 1:
The invention changes the temperature parameter from variable (PCR) to constant (isothermal), eliminating the need for temperature cycling hardware while maintaining amplification efficiency through optimized primer design and buffer conditions at a single temperature of 65°C
Solution Approach 2:
The invention extracts and removes the temperature cycling step from the amplification process, keeping only the essential isothermal extension step, thereby eliminating complex heating and cooling hardware while preserving the core amplification function
2Temperature
If LAMP method is used for nucleic acid amplification, then isothermal operation is achieved, but primer complexity increases
Solution Approach 1:
The invention segments the primer into two distinct functional regions: a 5' tail region for template binding and a 3' extension region for polymerase activity. This segmentation allows a single primer to perform multiple functions that traditionally required multiple primers, simplifying the primer design while maintaining isothermal operation
Solution Approach 2:
The invented primer design provides multi-functionality by enabling a single primer to both bind to the template (via the 5' tail) and serve as the extension site for polymerase (via the 3' end), replacing the need for separate binding and extension primers used in LAMP
3Quantity of substance
If conventional amplification methods are used, then multiple copies are generated, but process complexity increases
Solution Approach 1:
The invention performs preliminary action by designing primers with pre-configured 5' tail regions that are complementary to each other, so that after extension, the newly synthesized strands automatically contain the necessary binding sites for the next round of amplification, enabling self-sustaining exponential growth without complex procedural steps
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 amplifies nucleic acid templates, generating multiple copies while maintaining control over temperature, reducing the complexity of the amplification process and enhancing the yield of nucleic acid copies.
Implementation Method 1
treating a primary double-stranded nucleic acid comprising a 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 nucleic acid polymerases and primers.