Non-Isothermal Nucleic Acid Amplification for Point-of-Care Testing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing nucleic acid amplification techniques, such as PCR and isothermal methods like SDA and NEAR, are not suitable for point-of-care (PoC) applications due to the need for thermal cycling or constant temperature conditions, which are not practical outside laboratory settings.
Innovation Solution
A non-isothermal nucleic acid amplification method that involves hybridizing primers to a target sequence, creating nicking sites, and extending strands at varying temperatures, allowing for exponential amplification without thermal cycling, using a combination of thermophilic and mesophilic polymerases and nicking enzymes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If thermal cycling is used for PCR amplification, then amplification efficiency is improved, but device complexity and suitability for point-of-care applications deteriorates
Solution Approach 1:
The invention changes the temperature parameter from cyclic variation to a single sustained high temperature (65-75°C), eliminating the need for complex thermal cycling apparatus while maintaining amplification efficiency through the unique primer design and enzymatic reactions that function optimally at this constant temperature
2Quantity of substance
If high temperature (90°C) is used to dissociate double stranded target in SDA, then single stranded target availability is improved, but suitability for point-of-care applications deteriorates
Solution Approach 1:
The invention changes the temperature parameter from high temperature dissociation (90°C) to moderate temperature (65-75°C) by using primers with Tm values above the reaction temperature, allowing the target to remain partially double-stranded while still enabling primer binding and amplification through the nicking enzyme mechanism
3Device complexity
If isothermal amplification is used, then device complexity is reduced, but reaction time increases
Solution Approach 1:
The invention uses a sustained high temperature (65-75°C) that accelerates the nicking enzyme activity and polymerase extension rates, allowing the amplification reactions to proceed much faster than conventional isothermal methods while maintaining simple device requirements
4Ease of operation
If conventional isothermal amplification is used, then ease of operation is improved, but specificity decreases leading to non-specific amplification products
Solution Approach 1:
The invention introduces asymmetric nicking sites in the primers that create directional amplification, where the forward and reverse primers have different nicking site positions and sequences, allowing the nicking enzyme to selectively process only the intended target sequences and prevent non-specific amplification products
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 reduces reaction time, increases yield, and decreases non-specific amplification products, making it suitable for PoC devices without complex thermal cycling equipment.
Implementation Method 1
mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target
Implementation Method 2
causing a nick at each of said nicking sites in the strands of the duplex
Implementation Method 3
using a polymerase to extend the nicked strands so as to form newly synthesised nucleic acid
Data Source
Figure 1A~1C
Figure 2A
Figure 2A
AI summary
Disclosed is a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising the steps of: (a) mixing a target sequence with one or more complementary single stranded primers in conditions which permit a hybridisation event in which the primers hybridise to the target, which hybridisation event, directly or indirectly, leads to the formation of a duplex structure comprising two nicking sites disposed at or near opposite ends of the duplex; and performing an amplification process by; (b) causing a nick at each of said nicking sites in the strands of the duplex; (c) using a polymerase to extend the nicked strands so as to form newly synthesised nucleic acid, which extension with the polymerase recreates nicking sites; (d) repeating steps (b) and (c) as desired so as to cause the production of multiple copies of the newly synthesised nucleic acid; characterised in that the temperature at which the method is performed is non-isothermal, and subject to a reduction of at least 2º C during the amplification process of steps (b)-(d).