EM-SEq Nucleic Acid Amplification with Endonuclease-Mediated Shifting Equilibrium
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Solution Overview
Problem
Strand Displacement Amplification (SDA) methods face limitations due to high levels of dead-end product formation, which restricts exponential amplification and the ability to generate amplicons suitable for further sequencing, especially when performed on solid surfaces.
Innovation Solution
Endonuclease-Mediated Shifting Equilibrium Amplification (EM-SEq) introduces low melting point regions on nucleic acid strands to facilitate transient single-stranded hybridization with primers, allowing for snap extension and reducing dead-end product accumulation, thereby improving reaction kinetics and producing amplicons with regenerated endonuclease recognition sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Strand Displacement Amplification (SDA) is performed using conventional methods, then amplification can be achieved, but high levels of dead-end product formation occur which restricts exponential amplification and reduces amplification efficiency
Solution Approach 1:
The patent modifies the chemical parameters of the amplification reaction by introducing a branched DNA structure with a 3'-blocking group (such as a dideoxynucleotide or modified nucleotide) that prevents further extension. This parameter change in the primer structure allows the blocking group to be selectively removed by a specific endonuclease, thereby converting dead-end products into amplifiable products with free 3'-OH groups and restoring exponential amplification capability
Solution Approach 2:
The patent introduces a third enzyme component (a specific endonuclease such as a restriction enzyme or phosphatase) that acts as an intermediary to selectively process the blocking groups on dead-end products. This intermediary enzyme converts the non-amplifiable dead-end products back into amplifiable substrates without affecting the active amplification cycles, thereby resolving the accumulation of dead-end products and restoring exponential kinetics
2Ease of operation
If SDA is performed on solid surfaces, then spatial amplification is achieved, but the ability to generate amplicons suitable for further sequencing is reduced
Solution Approach 1:
The patent applies different functional properties to different parts of the amplification system: the solid surface provides localized amplification sites with immobilized primers, while the solution-phase branched DNA substrates and enzymes enable amplicon release with intact sequences. This local differentiation allows simultaneous achievement of spatial confinement benefits and sequencing compatibility
3Productivity
If conventional SDA is used, then amplification occurs, but off-target amplification increases due to temperature profile limitations
Solution Approach 1:
The patent employs dynamic temperature cycling combined with a three-enzyme system that operates at different temperature optima. The first enzyme (strand-displacing polymerase) operates at lower temperature for specific primer extension, while subsequent enzymes (endonuclease and ligase or second polymerase) operate at higher temperatures for processing and amplification. This dynamic temperature profile maintains high specificity while enabling exponential amplification rates
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
EM-SEq enhances the balance of products towards those with adapted ends, enabling efficient isothermal amplification and subsequent sequencing, while reducing the number of amplicon types and improving amplification efficiency on solid surfaces.
Implementation Method 1
modifying the ends of the strands in the population such that at least one of the ends contains a low melting point region of sequence which, at a temperature of above 37° C. and below 80° C., is at least transiently single stranded
Data Source
AI summary
The embodiments and improvements relate to the design of molecular biology assays based on isothermal amplification of nucleic acids with Strand Displacement Amplification (SDA). The embodiments describe a novel method for SDA termed Endonuclease-Mediated Shifting Equilibrium Amplification (EM-SEq), which improves exponential kinetics and specificity of the reaction and enables amplification on solid surfaces.