Denaturation Bubble-Mediated Strand Exchange Amplification
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Solution Overview
Problem
Current nucleic acid amplification technologies, such as PCR and isothermal amplification methods, face limitations in speed and throughput, with conventional PCR being slow due to thermal cycler constraints and isothermal methods prone to contamination and non-specific amplification.
Innovation Solution
The denaturation bubble-mediated strand exchange amplification (SEA) method, which utilizes spontaneous denatured regions in DNA to facilitate amplification without thermal cyclers, by subjecting a sample to thermal cycles between specific temperatures to enhance amplification efficiency and speed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional PCR is used to amplify nucleic acids, then amplification sensitivity is achieved, but amplification speed is slow due to thermal cycler limitations
Solution Approach 1:
The patent replaces the mechanical thermal cycler system with a chemical denaturation bubble-mediated system. Instead of using mechanical heating and cooling cycles to denature and anneal DNA, the invention uses spontaneously formed denaturation bubbles in dsDNA at constant temperature, allowing primer invasion and strand exchange without thermal cycling equipment.
Solution Approach 2:
The patent changes the temperature parameter from dynamic cycling to constant temperature. By maintaining the reaction at a single optimal temperature (e.g., 65°C for Bst polymerase), the system eliminates the time-consuming thermal transitions while preserving amplification functionality through temperature-independent denaturation bubbles.
2Speed
If thermal cyclers are used to reduce reaction volume for faster energy transfer, then amplification speed increases, but device complexity and cost increase
Solution Approach 1:
The patent extracts and eliminates the thermal cycler instrument from the amplification system. By removing the need for programmed temperature cycling equipment, the invention simplifies the device complexity while maintaining amplification speed through the constant-temperature denaturation bubble mechanism.
Solution Approach 2:
The patent enables the DNA template to self-denature through spontaneously formed denaturation bubbles at constant temperature, eliminating the need for external thermal cycling control. The system serves itself by utilizing intrinsic DNA properties rather than requiring complex instrument intervention.
3Ease of operation
If isothermal amplification methods are used to simplify the process, then ease of operation improves, but contamination risk and non-specific amplification increase
Solution Approach 1:
The patent introduces denaturation bubbles as an intermediary mechanism that mediates between the constant temperature condition and specific amplification. These bubbles serve as controlled entry points for primers, enabling specific primer-template binding while preventing non-specific interactions, thus maintaining reliability under isothermal conditions.
Solution Approach 2:
The patent introduces dynamic denaturation bubbles that continuously form and dissolve in the dsDNA template at constant temperature. This dynamic process allows controlled access to single-stranded regions for specific primer binding, while the transient nature of bubbles prevents stable non-specific binding, thereby maintaining amplification specificity.
4Speed
If rapid heating methods like infrared lamps or microwaves are used to accelerate amplification, then amplification speed increases, but heating accuracy and sensitivity decrease
Solution Approach 1:
The patent replaces rapid mechanical heating methods with a chemical/biological mechanism using denaturation bubbles. Instead of applying external energy fields that lack temperature control, the system uses spontaneous DNA breathing and bubble formation to achieve denaturation with inherent precision at constant temperature.
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 significantly accelerates nucleic acid amplification by thousands of folds, offering a high-throughput and stable solution suitable for laboratory use, while eliminating the need for thermal cyclers and reducing contamination risks.
Implementation Method 1
subjecting the amplification mixture to a number of thermal cycles between a first temperature and a second temperature
Implementation Method 2
utilizes spontaneous denatured regions in DNA to facilitate amplification
Implementation Method 3
amplifying a sequence of the target nucleic acid molecule through polymerase chain reaction (PCR)
Data Source
AI summary
Provided herein is a method for denaturation bubble-mediated target nucleic acid amplification and related kits and uses thereof. The method facilitates the generation of denaturation bubbles in a duplex target nucleic acid molecule through the application of swift temperature changes during a thermal cycle, thereby accelerating the strand exchange amplification (SEA) reaction. The kits comprise specially designed primers and polymerase configured for performing the method. The methods and kits disclosed herein can be used under various scenarios, such as diagnosis of infectious or genetic diseases, sample quality control, and single nucleotide polymorphism (SNP) profiling.