Reversibly Inactivated Nuclease for Nucleic Acid Contamination Control
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Solution Overview
Problem
Existing nucleic acid amplification techniques face challenges in effectively eliminating synthesized nucleic acid post-reaction to prevent contamination in subsequent assays, particularly in isothermal amplification methods like STAR and qSTAR, where temperature deviations are required, and in reducing the risk of false positive results due to residual amplifiable fragments.
Innovation Solution
A method involving the use of a temporarily substantially inactive nuclease, which regains activity after a sufficient period to digest in vitro synthesized nucleic acid, allowing for its detection and subsequent degradation, thereby reducing contamination risks. This is achieved by initially inactivating the nuclease using reducing agents and controlled temperature conditions, ensuring the nuclease remains inactive during synthesis and becomes active post-detection for digestion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a nuclease is permanently inactivated to prevent contamination of synthesized nucleic acid, then contamination risk is reduced, but the ability to digest nucleic acid when needed is lost
Solution Approach 1:
The nuclease activity is made dynamic through reversible inactivation. The system transitions between active and inactive states based on reaction phase: inactive during synthesis to prevent contamination, and active during cleanup to digest products. This is achieved through chemical modifiers that can be added or removed to control nuclease state, enabling the system to adapt to different operational requirements.
Solution Approach 2:
The nuclease activity is controlled by changing chemical parameters - specifically the presence or absence of modifying agents like DTT or iodoacetamide. These agents alter the nuclease's functional state without permanently destroying it. By adjusting these chemical parameters, the system can switch between states suitable for different phases of the nucleic acid synthesis and cleanup process.
2Productivity
If the nuclease remains active throughout the synthesis reaction, then digestion of synthesized nucleic acid is immediate, but the synthesized nucleic acid is degraded during the reaction
Solution Approach 1:
The nuclease is prepared in an inactive state before the synthesis reaction begins. This preliminary inactivation prevents the nuclease from degrading the synthesized nucleic acid during the reaction. The activation to enable digestion occurs only after synthesis is complete, ensuring that the nucleic acid product is fully formed and detectable before being subjected to digestion.
Solution Approach 2:
The nuclease activity is applied periodically rather than continuously - inactive during the synthesis phase and active during the cleanup phase. This periodic control allows the system to benefit from nuclease digestion at the appropriate time while avoiding degradation during synthesis, optimizing both productivity and substance preservation.
3Reliability
If reducing agents like DTT are used to inactivate the nuclease, then nuclease activity is suppressed during synthesis, but residual nuclease activity may remain
Solution Approach 1:
The harmful residual nuclease activity is removed by adding an excess of modifying agent (such as iodoacetamide) that reacts with and permanently inactivates any remaining active nuclease molecules. This extraction of harmful activity ensures complete suppression of nuclease function during the synthesis phase, eliminating the risk of degradation while maintaining the ability to reactivate sufficient nuclease for later cleanup.
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
The method effectively digests synthesized nucleic acid, reducing the risk of contamination and allowing for the reuse of equipment in nucleic acid amplification reactions, while maintaining assay performance by incorporating reversibly inactivated nucleases into the reaction mixture, ensuring complete or partial digestion of amplification products within 24 to 48 hours post-reaction.
Implementation Method 1
permitting or causing the substantially inactive nuclease to regain substantial nuclease activity after a period of time has elapsed sufficient to allow detection of the in vitro synthesised nucleic acid, such that the in vitro synthesised nucleic acid is digested by the nuclease
Implementation Method 2
The preferred treatment required to cause reversible inactivation of the nuclease will depend on the identity of the nuclease concerned. For example, HL-SAN and HL-dsDNase (both from ArcticZymes), are described by the manufacturer as being irreversibly inactivated by the presence of 1 mM DTT
Data Source
AI summary
Disclosed herein is a method of causing enzymatic digestion of in vitro synthesised nucleic acid (especially DNA), the method comprising the steps of: (a) combining reagents, in the presence of a temporarily substantially inactive nuclease, to form in vitro synthesised nucleic acid; and (b) subsequently permitting or causing the substantially inactive nuclease to regain substantial nuclease activity after a period of time has elapsed sufficient to allow detection of the in vitro synthesised nucleic acid, such that the in vitro synthesised nucleic acid is digested by the nuclease.