Shewanella Phage Nickase for Isothermal Amplification
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Solution Overview
Problem
Current methods for nucleic acid detection, such as strand displacement amplification, require costly and cumbersome procedures due to the limited availability of nickase-producing bacteria and restricted genetic modification scope, hindering widespread application in fields like food safety and biomedical testing.
Innovation Solution
Shewanella oneidensis MR-1 bacteria, deposited at ATCC 700550, is found to produce a nickase encoded by a temperate phage fragment, providing a new source for nickase-producing bacteria with a wide range of action temperatures (4 to 55°C) and improved cutting performance, enabling more efficient nucleic acid isothermal amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If HincII is used for DNA cutting, then double-strand breaks are generated, but this leads to high cost and cumbersome procedure requiring modified substrates
Solution Approach 1:
The patent extracts and utilizes the nickase function from the phage fragment genome of Shewanella oneidensis MR-1, separating the desired single-strand cutting activity from the complexity of modified substrate requirements. By identifying and expressing the nickase gene (SO_0655) from the phage fragment, the invention obtains a enzyme that naturally performs single-strand cutting without requiring dATPoS modification, thus simplifying the experimental procedure while maintaining detection reliability
Solution Approach 2:
The patent changes the enzymatic parameter from double-strand cutting (HincII) to single-strand cutting (nickase), fundamentally altering the reaction mechanism. This parameter change eliminates the need for modified substrates and complex procedures, as the nickase naturally recognizes and cuts single strands at specific sequences without requiring additional chemical modifications
2Ease of operation
If nickase is used to simplify procedures, then sample contamination is reduced, but limited nickase-producing bacteria restricts application scope
Solution Approach 1:
The patent discovers and characterizes a novel nickase from Shewanella oneidensis MR-1 that exhibits broad temperature adaptability (4-55°C), making it universally applicable across different experimental conditions. This single enzyme source can replace multiple temperature-specific nickases, expanding the versatility of nickase applications while maintaining procedural simplicity and reducing contamination risks
Solution Approach 2:
The patent utilizes the bacterium's natural capability to produce nickase as part of its normal metabolism or stress response, rather than requiring complex genetic engineering to introduce foreign genes. The Shewanella oneidensis MR-1 strain naturally expresses the nickase activity, allowing for straightforward cultivation and enzyme extraction, thus simplifying the production process while expanding available sources
3Reliability
If modified substrate dATPoS is introduced to enable single-strand cutting, then nickase activity is achieved, but cost increases significantly
Solution Approach 1:
The patent extracts the essential nickase cutting function from the complex modified substrate system. By identifying and purifying the nickase enzyme directly from Shewanella oneidensis MR-1, the invention eliminates the need for expensive dATPoS modified substrates. The enzyme itself provides the specificity for single-strand cutting without requiring additional chemical modifications, thus maintaining reliability while dramatically reducing reagent costs
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 nickase derived from Shewanella oneidensis MR-1 offers enhanced enzyme activity and broader application values, simplifying experimental procedures and reducing sample contamination, thus facilitating rapid and efficient nucleic acid detection.
Implementation Method 1
Nickase (Nicking nuclease) can effectively replace DNA double-strand endonuclease for a rapid constant temperature amplification technology
Implementation Method 2
Strand displacement amplification (SDA) was proposed by Walker et al. in 1992 for the rapid detection of Mycobacterium tuberculosis IS6110 nucleic acid fragments
Data Source
AI summary
A nickase encoded by Shewanella integrated temperate phage fragments and an application thereof are provided. The nickase is derived from Shewanella oneidensis MR-1 (ATCC 700550), the amino acid sequence of the nickase is as shown in SEQ ID NO: 2, and the nucleotide sequence of the nickase is as shown in SEQ ID NO: 1. It can break a specific part of one of single strands of double-stranded DNA. The nickase has a wide range of action temperatures and good cutting effects, and has wide application values in the fields of nucleic acid isothermal amplification and the like.