Methylation-Protected DNA Assembly for Scarless Large Constructs
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Solution Overview
Problem
Existing methods for DNA assembly face challenges in achieving efficient assembly of large and arbitrary DNA sequences without leaving unwanted scar sequences and designing methods, particularly in the context of existing technologies are not effective for designing and assembling large DNA constructs, and existing methods are not effective for assembling large and arbitrary DNA sequences without leaving unwanted scar sequences in the final assembled DNA, and existing methods are not effective for assembling large and arbitrary DNA sequences without causing a failure in the cloning process, and existing methods are not effective for assembling large and arbitrary DNA sequences without making custom assembly vectors, and existing methods are not efficient for assembling large and arbitrary sequences of DNA sequences without addressing the need for assembling large and arbitrary sequences without making custom assembly vectors.
Innovation Solution
A DNA assembly method using a single restriction enzyme with methylation-protectable restriction elements that allow for scarless assembly by blocking the overlapping restriction enzyme recognition site, enabling the use of a single enzyme throughout different stages of DNA assembly, providing more freedom in adapter sequence design, and allowing for hierarchical assembly of large and arbitrary DNA sequences without unwanted scar sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If type IIS restriction enzyme-based assembly methods are used, then assembly modularity and part reusability are improved, but unwanted scar sequences are introduced and sequence design constraints increase
Solution Approach 1:
The patent extracts and removes the type IIS restriction enzyme recognition sequences from the final assembled DNA construct through precise adapter design. The adapters are engineered to contain restriction sites that are present in the assembly intermediates but are excluded from the final product, effectively taking out the harmful scar sequences while maintaining the benefits of modular assembly.
Solution Approach 2:
The patent applies preliminary action by pre-designing adapters with embedded restriction enzyme recognition sequences before assembly. These adapters facilitate the assembly process by providing defined sticky ends, and the restriction sites are subsequently removed through selection strategies, allowing the assembly to proceed smoothly while planning for scar removal in advance.
2Length of moving object
If multiple restriction enzymes are used for hierarchical assembly, then assembly of large DNA fragments is enabled, but device complexity and cost increase
Solution Approach 1:
The patent applies universality by designing a system where a single type IIS restriction enzyme can be used for multiple assembly stages. The adapters are engineered to contain the same restriction enzyme recognition sequence, allowing the same enzyme to cut and ligate fragments at different hierarchical levels, thereby reducing device complexity while maintaining the ability to assemble large DNA fragments.
3Object-generated harmful factors
If restriction enzyme recognition sequences are removed from assembled DNA, then scarless assembly is achieved, but assembly modularity and reusability are reduced
Solution Approach 1:
The patent uses preliminary action by incorporating restriction enzyme recognition sequences into adapter regions that are designed to be present during assembly but removed in the final product. The adapters serve their function of facilitating modular assembly with defined sticky ends, and the embedded restriction sites are subsequently excluded through selection strategies, achieving both scarless assembly and maintained reusability.
Solution Approach 2:
The patent employs adapters as intermediary elements that contain the restriction enzyme recognition sequences. These adapters mediate the assembly process by providing compatible sticky ends for ligation, and the restriction sites within the adapters serve as markers for subsequent selection steps that remove the intermediaries, leaving no scars in the final construct while the adapter design principles can be reused.
4Productivity
If internal restriction sites are removed from DNA parts, then assembly efficiency is improved, but manufacturing complexity and cost increase
Solution Approach 1:
The patent extracts internal restriction sites from DNA parts through adapter-mediated assembly. The adapters are designed to ligate to the DNA parts and provide external restriction sites that replace the internal ones. This taking out of problematic internal sites improves assembly efficiency by preventing unwanted cuts, while the adapter design and selection strategies streamline the manufacturing process rather than complicating it.
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 enables efficient assembly of large and arbitrary DNA sequences with minimal scarring, reducing the complexity and cost of DNA assembly by using a single enzyme, and allowing for the assembly of large DNA constructs with repetitive sequences without leaving unwanted scar sequences, thus overcoming the limitations of existing methods.
Implementation Method 1
a base within the restriction enzyme recognition sequence is modified by the DNA methylase
Data Source
AI summary
The invention relates to a nucleic acid for use in DNA assembly, wherein the nucleic acid comprises at least one methylation-protectable restriction element, the methylation-protectable restriction element comprising: a restriction enzyme recognition sequence that is recognised by a restriction enzyme that cleaves outside of the recognition sequence; and a DNA methylase recognition sequence, wherein the restriction enzyme recognition sequence and the DNA methylase recognition sequence overlap such that the base modified by the DNA methylase lies within the restriction enzyme recognition sequence, wherein the DNA methylase recognition sequence is not identical to or enclosed by the restriction enzyme recognition sequence, and wherein the DNA methylase recognition sequence does not overlap with the sequence that would form the overhang end sequence generated by the restriction enzyme. The invention further relates to asscociated methods and kits.