Type IIs Enzyme DNA Vector Assembly
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Solution Overview
Problem
Current DNA cloning methods are inefficient and error-prone, requiring multiple steps, multiple enzymes, and often result in residual sequences or 'scar' nucleotides in the final vector, limiting control over vector composition and compatibility between different plasmids.
Innovation Solution
A method using a single type IIs restriction enzyme to assemble linear double-stranded DNA molecules with sequences of interest flanked by double-stranded DNA adapters, eliminating enzyme recognition sites and forming cohesive single-stranded sutures for precise vector assembly without multiple cloning sites or residual sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional restriction enzyme and ligase methods are used for DNA cloning, then DNA fragments can be inserted into vectors, but multiple steps and multiple enzymes are required, increasing the risk of contamination and errors
Solution Approach 1:
The patent combines the functions of multiple restriction enzymes into a single type IIs restriction enzyme system. This enzyme simultaneously performs recognition, cleavage, and generation of compatible cohesive ends for multiple DNA fragments in one reaction, eliminating the need for sequential enzyme treatments and reducing contamination risks
Solution Approach 2:
The type IIs restriction enzyme system is designed to be universal, working with various DNA sequences and vectors through standardized adapter designs. The enzyme system can assemble multiple different DNA fragments simultaneously using the same enzymatic reaction conditions, making the method applicable to diverse cloning scenarios without requiring enzyme optimization for each case
2Ease of manufacture
If multiple restriction enzymes are used for DNA assembly, then specific insertion sites can be targeted, but the presence or absence of restriction sites in plasmid sequences makes sequence transfers complex
Solution Approach 1:
The patent introduces standardized DNA adapters as intermediary elements between the type IIs restriction enzyme and the target DNA sequences. These adapters contain the recognition sites for the restriction enzyme and provide uniform cohesive ends, mediating the connection between diverse DNA fragments and vectors without requiring the vectors themselves to contain specific restriction sites
Solution Approach 2:
The method changes the fundamental parameter of how DNA fragments are connected by using type IIs restriction enzymes that cleave outside their recognition sites. This generates standardized cohesive ends regardless of the internal sequence composition of the DNA fragments, allowing seamless assembly of sequences that would otherwise be incompatible with traditional restriction-based cloning
3Adaptability or versatility
If ligation-independent cloning or Gibson Assembly is used, then multiple restriction enzymes are avoided, but control over the functionalities of the final vector is limited
Solution Approach 1:
The patent segments the DNA assembly process into modular components: type IIs restriction enzyme for cleavage, standardized adapters for connection, and controllable functional elements. This segmentation allows independent optimization and selection of each component, providing full control over vector functionalities while maintaining ease of manufacture through standardized protocols
4Reliability
If traditional cloning methods with multiple steps are used, then DNA fragments can be assembled, but the number of steps increases the risk of exposure to exogenous contaminants that could degrade the DNA
Solution Approach 1:
The patent performs preliminary action by designing DNA fragments with pre-attached adapters containing type IIs restriction sites before the assembly reaction. This pre-preparation allows all fragments to be ready for simultaneous cleavage and ligation in a single reaction step, reducing the time DNA is exposed to potentially contaminating enzymes and buffers while maintaining high assembly efficiency
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 the rapid, cost-effective, and error-free assembly of custom expression vectors with total control over sequence composition, eliminating the need for multiple enzymes and reducing production time and costs.
Implementation Method 1
a) a step of simultaneously contacting at least two molecular building blocks, which are different from one another, in the presence of a single restriction enzyme, said single restriction enzyme being a type IIs restriction enzyme... to the elimination by cleaving of the recognition sites of the type IIs restriction enzyme used
Implementation Method 2
to the pairing by nucleotide complementarity of the aforementioned cohesive single-stranded sutures of at least 2 nucleotides
Implementation Method 3
b) a step of ligation of the aforementioned cohesive single-stranded sutures of at least 2 nucleotides, so as to obtain a circular double-stranded DNA vector
Data Source
AI summary
Disclosed is a method for producing, in one step, “made to measure” double-stranded DNA vectors from molecular bricks including sequences of interest in the presence of a one and only type IIs restriction enzyme.