Isothermal Nucleic Acid Amplification via Strand-Displacement Polymerase
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Solution Overview
Problem
Current nucleic acid amplification methods under isothermal conditions require complex primer designs and additional enzymes, leading to increased costs and longer reaction times, while traditional PCR methods face challenges with temperature control and efficiency.
Innovation Solution
A nucleic acid amplification method using oligonucleotide primers designed to target specific sequences within 200 nucleotides, allowing for efficient amplification at room temperature to 100°C with DNA polymerase having strand displacement activity, eliminating the need for restriction enzymes and complex primer designs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional PCR method is used to perform nucleic acid amplification, then amplification efficiency is improved, but temperature control becomes complicated and reaction time increases
Solution Approach 1:
The invention changes the temperature parameter from variable (PCR method requiring multiple temperature steps) to constant (isothermal condition at 60-70°C), thereby simplifying the temperature control system while maintaining amplification efficiency through the use of strand-displacement-type DNA polymerase
2Device complexity
If isothermal amplification methods such as LAMP or ICAN are used, then temperature control is simplified, but primer design becomes very difficult and additional enzymes are required
Solution Approach 1:
The invention extracts and eliminates the requirement for complex primer designs and additional enzymes (such as restriction enzymes in SDA or multiple primers in LAMP) by using a simple pair of primers with strand-displacement-type DNA polymerase, thereby maintaining isothermal simplicity while reducing primer design difficulty
Solution Approach 2:
The strand-displacement-type DNA polymerase performs multiple functions: it extends primers, displaces complementary strands, and enables isothermal amplification without requiring additional enzymes or complex primer structures, thereby simplifying the overall system
3Temperature
If SDA method using restriction enzyme and DNA polymerase is used, then isothermal amplification is achieved, but cost increases and primer design requires improvement
Solution Approach 1:
The invention extracts and removes the requirement for restriction enzymes from the amplification system, using only strand-displacement-type DNA polymerase and simple primers, thereby reducing reagent costs while maintaining isothermal amplification capability
Solution Approach 2:
The invention replaces expensive restriction enzymes with a more cost-effective DNA polymerase system that achieves the same strand displacement function, making the method more economically viable for routine applications
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 achieves high-efficiency nucleic acid amplification in a shorter time with simplified primer design, reducing costs and reaction time, and maintaining high specificity and sensitivity.
Implementation Method 1
nucleic acid amplification is generally performed by an enzymatic method using DNA polymerase. Polymerase chain reaction (PCR) is broadly known as a nucleic acid amplification method.
Implementation Method 2
using a strand-displacement-type DNA polymerase lacking 5'→3' exonuclease activity and synthesizing a complementary strand while displacing a complementary strand
Implementation Method 3
annealing (annealing step) primers to the single-stranded DNAs
Data Source
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Figure 3
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AI summary
An object to be achieved by the present invention is to provide a nucleic acid amplification method by which a nucleic acid can be amplified using oligonucleotide primers and DNA polymerase. The present invention provides a nucleic acid amplification method which comprises performing incubation of a reaction solution containing at least one type of deoxynucleotide triphosphate, at least one type of DNA polymerase having strand displacement activity, at least two types of oligonucleotide primer, and the nucleic acid fragment as a template so as to perform a polymerase reaction that initiates from the 3' end of the primer and thus amplifying the nucleic acid fragment, wherein a first oligonucleotide primer and a second oligonucleotide primer are designed in such a way that a region which contains two identical sequences X of serial 4 or more nucleotides within the region of 200 or less nucleotides, or a part thereof can be amplified.