Dry DNA Assembly on Filter Paper Carriers
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Solution Overview
Problem
Current DNA assembly methods are costly and inefficient, particularly for large-scale storage and cloning, as they require low-temperature storage of purified plasmid DNA, which is expensive and prone to quality deterioration, and involve labor-intensive processes for DNA extraction and bacterial transformation.
Innovation Solution
A method for one-pot, one-step assembly of DNA molecules using dry DNA aliquots flanked by restriction endonuclease type IIS interfaces on a carrier, such as filter paper or cellulose particles, which can be stored at room temperature and directly assembled in a microreaction vessel with enzymes and buffers, eliminating the need for bacterial transformation and DNA extraction.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If purified plasmid DNA is stored at low temperature (-20°C), then DNA stability is improved, but storage cost increases and quality deterioration still occurs over time
Solution Approach 1:
The patent changes the physical state parameter of DNA from dissolved (requiring cold storage) to dried state (stable at room temperature). By removing water through drying, the DNA achieves long-term stability without requiring low-temperature storage, thereby eliminating freezer infrastructure costs while maintaining DNA integrity
Solution Approach 2:
The patent employs disposable filter paper carriers that can be directly used in assembly reactions without requiring DNA extraction. These single-use carriers eliminate the need for expensive cold storage infrastructure and repeated DNA purification steps, reducing both capital and operational costs
2Reliability
If purified plasmid DNA is stored at low temperature, then DNA quality is maintained, but the process becomes labor-intensive requiring extraction and transformation
Solution Approach 1:
The patent performs preliminary drying of DNA on filter paper carriers before storage, creating a stable, room-temperature-storable format. This preliminary action eliminates the need for subsequent DNA extraction steps and cold storage maintenance, simplifying the workflow while preserving DNA quality through the drying process
Solution Approach 2:
The patent extracts DNA from the aqueous plasmid preparation context and immobilizes it on dry filter paper carriers. This extraction from the traditional dissolved state eliminates the requirement for cold storage and complex extraction protocols during use, as the DNA remains accessible on the carrier throughout storage and assembly
3Manufacturing precision
If traditional cloning methods are used with multiple steps, then assembly precision can be maintained, but productivity decreases due to multiple cloning steps
Solution Approach 1:
The patent merges multiple sequential cloning steps into a single one-pot assembly reaction. DNA fragments dried on different filter paper carriers are combined with enzymes and buffer in one reaction vessel, allowing simultaneous assembly of multiple fragments while maintaining precision through standardized carrier-based delivery
Solution Approach 2:
The filter paper carrier serves multiple functions: DNA immobilization, storage, direct reaction template, and easy separation medium. This universal carrier system enables both precise assembly through standardized interfaces and high productivity through single-step reactions that eliminate intermediate purification steps
Data Source
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AI summary
The invention relates to a method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule, and a substrate and a kit for this purpose. The object of the invention is to provide a simple and cost-effective assembly method for DNA molecules. In order to achieve this object, a method for one-pot one-step assembly of two or more DNA molecules to form at least one recombinant DNA molecule is provided according to the invention, wherein the two or more DNA molecules to be assembled are brought together in dry form with a suitable reaction medium on at least one substrate present in a reaction vessel.