Modular Cloning System Using Type IIs Restriction Endonucleases
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Solution Overview
Problem
Current cloning methods for recombinant DNA molecules are slow, labor-intensive, and limited in assembling large or complex DNA constructs, as they require construct-specific strategies and are not well-suited for combinatorial assembly of multiple genetic elements.
Innovation Solution
A system using type IIs restriction endonucleases that allows assembly of multiple DNA fragments in a single step with a fixed set of cloning vectors, enabling the reuse of vectors for successive cloning steps and the creation of increasingly complex constructs without the need for construct-specific strategies.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional type II restriction enzymes and ligase are used for constructing recombinant DNA molecules, then the method is versatile, but the process becomes slow and labor-intensive, and only allows creation of constructs of relatively small size
Solution Approach 1:
The patent combines multiple DNA fragments (at least three fragments) into a single recombinant DNA molecule in one ligation reaction, rather than assembling them sequentially. This merging approach allows simultaneous assembly of multiple fragments with different sequences, significantly increasing productivity while maintaining versatility through the use of standardized overhangs that can accommodate various genetic elements.
2Adaptability or versatility
If all available restriction enzymes are used to design cloning strategies for large constructs, then comprehensive coverage is achieved, but the design becomes extremely difficult since all restriction enzymes will cut many times in such constructs
Solution Approach 1:
The patent extracts the cloning process from dependence on restriction enzyme recognition sites by using synthetic single-stranded overhangs instead. This extraction eliminates the problem of multiple cutting sites in large constructs, as the overhangs are designed specifically for the desired assembly rather than being determined by natural restriction sites that would cut throughout the construct.
Solution Approach 2:
The patent introduces synthetic single-stranded overhangs as intermediaries between DNA fragments to be assembled. These overhangs serve as mediators that facilitate specific annealing and ligation of fragments without requiring restriction enzyme recognition sites, thereby simplifying the cloning strategy design for large constructs while maintaining comprehensive adaptability.
3Productivity
If recombinase-based cloning is used to eliminate multiple occurrence of restriction sites, then the problem of restriction site multiplicity is solved, but recombination sites are left in the final construct, preventing seamless assembly of protein coding sequences
Solution Approach 1:
The patent extracts the assembly mechanism from recombination-based methods and uses ligation of fragments with complementary single-stranded overhangs instead. This extraction allows the assembly process to proceed without leaving recombination sites in the final construct, thereby maintaining seamless protein coding sequences while achieving efficient assembly of multiple fragments.
4Adaptability or versatility
If ligation-independent cloning is used to avoid restriction sites, then independence from restriction sites is achieved, but PCR is required and sequencing of constructs is necessary
Solution Approach 1:
The patent combines the advantages of restriction-site independence with traditional ligation-based cloning by using synthetic single-stranded overhangs that enable direct ligation without PCR amplification or sequencing verification. This merging approach maintains independence from restriction sites while eliminating the additional complexity of PCR and sequencing steps.
Data Source
AI summary
System for producing a nucleic acid construct of interest, said system comprising:a set of n entry DNAs numbered 1 to n, n being an integer of at least 2,each of said n entry DNAs comprising in this order:(i) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(ii) a sequence portion linking the cleavage site of said recognition site of item (i) with the cleavage site of the recognition site of the following item (iii), and(iii) a cleavage site of a further type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site;the cleavage sites of the type IIs restriction endonuclease recognition sites of item (iii) of entry DNAs 1 to n−1 are complementary to the cleavage sites of the type IIs restriction endonuclease recognition sites of item (i) of entry DNAs 2 to n, respectively;the cleavage site of the type IIs restriction endonuclease recognition site of item (iii) of entry DNA n is complementary to the cleavage site of the type IIs restriction endonuclease recognition site of item (i) of entry DNA 1 for allowing annealing of complementary single-stranded overhangs formed by restriction at recognition site (i) of entry DNA 1 and at recognition site (iii) of entry DNA n;said system further comprising a destination vector comprising in this order:(I) a type IIs restriction endonuclease recognition site followed by the cleavage site thereof;(II) a vector backbone preferably comprising a selectable marker gene, said vector backbone linking the cleavage sites of said recognition sites of items (I) and the following item (III);(III) a further cleavage site of a type IIs restriction endonuclease recognition site followed by the recognition site of said cleavage site, and(IV) optionally, an insert between the recognition sites of item (III) and item (I);said cleavage sites of items (I) and (III) being different and non-complementary, said recognition sites of items (I) and (III) being preferably recognitions sites of the same endonuclease.