Isothermal Strand Displacement Amplification Without Denaturation
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Solution Overview
Problem
Current isothermal strand displacement amplification methods require denaturation of double-stranded DNA to facilitate primer binding, which limits their specificity and efficiency, especially in biological samples where natural nicking sites may not be present or accessible.
Innovation Solution
The development of primers with modified bases and a 5′-non-complementary tail containing a nicking enzyme-specific sequence allows for efficient primer extension and amplification without denaturation, utilizing a nicking enzyme to create a nicking site during amplification, enabling specific amplification of double-stranded or single-stranded nucleic acid targets at isothermal conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If heat denaturation at 95°C is applied to separate double-stranded DNA into single strands, then primer binding is enabled, but amplification specificity and efficiency deteriorate due to non-specific binding and loss of target integrity
Solution Approach 1:
The patent changes the fundamental parameter of DNA strand separation from thermal denaturation (95°C) to enzymatic nicking at isothermal conditions (37-65°C). This allows primer binding without the harmful effects of heat denaturation, maintaining target integrity and amplification specificity while enabling efficient primer extension through controlled enzymatic action
Solution Approach 2:
The patent replaces the mechanical/thermal system (heat denaturation) with a biochemical system (nicking enzyme-mediated strand separation). The nicking enzyme creates controlled nicks in one strand of the double-stranded DNA, allowing primer binding and extension without the need for high-temperature denaturation, thus resolving the contradiction between ease of operation and reliability
2Productivity
If hemiphosphorothioate sites are generated to enable nicking by restriction enzymes, then strand displacement amplification is achieved, but device complexity and manufacturing difficulty increase due to specialized reagent preparation
Solution Approach 1:
The patent extracts the hemiphosphorothioate modification step from the amplification system, using instead naturally occurring nicking enzymes that recognize and cleave specific DNA sequences without requiring chemically modified substrates. This simplifies reagent preparation while maintaining strand displacement amplification efficiency through the natural enzymatic activity of nicking enzymes
Solution Approach 2:
The patent employs commercially available nicking enzymes with defined recognition sequences that can be used directly in the amplification reaction without complex pre-preparation. These enzymes are stable, easy to handle, and do not require the sophisticated reagent preparation involving hemiphosphorothioate site generation, thus improving ease of manufacture while maintaining productivity
3Reliability
If a limited number of nicking enzymes are used with specific recognition sequences, then specific nicking sites are created, but adaptability deteriorates when no natural nicking site is present in the target region
Solution Approach 1:
The patent segments the primer into two functional parts: a 5' non-complementary tail containing the nicking enzyme recognition sequence and a 3' complementary region that binds to the target. This segmentation allows the nicking site to be artificially introduced at any target location through primer design, combining nicking precision with universal target applicability
Solution Approach 2:
The patent performs preliminary action by incorporating the nicking enzyme recognition sequence into the 5' tail of the primer before the amplification reaction begins. This pre-positioning of the nicking site ensures that the nicking enzyme can act at the desired location during amplification, enabling both specific nicking and broad target applicability without requiring natural nicking sites
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 enables specific and efficient amplification of nucleic acid targets in biological samples without the need for denaturation, improving sensitivity and specificity, and allowing for detection using various state-of-the-art methods such as fluorescence resonance energy transfer (FRET) or lateral flow assays.
Implementation Method 1
a nicking enzyme to create a nicking site during amplification
Implementation Method 2
Watson-Crick pairs in naked DNA spontaneously flip into Hoogstein pairs under ordinary conditions, suggesting that DNA breathes
Data Source
AI summary
Methods, primers and probes are provided for the isothermal amplification and detection, without denaturation, of double stranded nucleic acid targets for polymerase strand displacement amplification (“iSDA”). The methods and compositions disclosed are highly specific for nucleic acid targets with high sensitivity, specificity and speed that allow detection of clinical relevant target levels. The methods and compositions can easily be used to amplify or detect nucleic acid targets in biological samples.