Intron-Based Universal Cloning via Type IIs Restriction
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Solution Overview
Problem
Current molecular cloning methods require unique and compatible prefix and suffix sequences for DNA modules, limiting the universal applicability and efficiency in assembling genetic components, especially in type IIs-based restriction-ligation reactions, and there is a need for methods to generate generic DNA fragment modules that can be reused independently of their 5′ and 3′ sequences.
Innovation Solution
A method involving the use of type IIs restriction endonucleases and ligases to fuse polynucleotides with intron sequences, allowing for the production of fusion polynucleotides encoding polypeptides by cleaving and ligating DNA fragments with complementary intron ends, enabling the assembly of DNA fragments without the need for compatible prefix and suffix sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If type IIs restriction-ligation is used to assemble DNA fragments, then assembly efficiency is improved, but the requirement for unique and compatible prefix and suffix sequences limits universal applicability
Solution Approach 1:
The intron-based cloning system provides a universal platform where all DNA fragments use the same intron sequences (5' intron and 3' intron) regardless of their specific identity. This allows any combination of DNA fragments to be assembled together using the same type IIs restriction enzyme and ligase, making the system universally applicable across different cloning projects without requiring fragment-specific optimization
Solution Approach 2:
The invention changes the cloning parameters by replacing the traditional requirement for unique prefix/suffix sequences with standardized intron sequences that contain type IIs restriction sites. By modifying the cloning strategy to use intron-based assembly with universal sequences, the system achieves both high productivity through efficient one-pot reactions and broad versatility through universal applicability
2Manufacturing precision
If multiple cloning steps are used with different type II restriction enzymes, then directional assembly is achieved, but compatibility issues between enzymes increase complexity
Solution Approach 1:
The invention merges multiple cloning steps into a single one-pot reaction by combining the type IIs restriction enzyme digestion and DNA ligase assembly steps. This is achieved by using intron sequences with type IIs restriction sites that are cleaved by the same enzyme, generating compatible overhangs that can be ligated in a single reaction mixture, thereby eliminating the need for sequential cloning steps with different enzymes
Solution Approach 2:
The type IIs restriction enzyme system provides universal functionality for both directional cleavage and fragment assembly. The same enzyme can cleave different intron sequences at specific positions to generate directional overhangs, and the same ligase can assemble any combination of cleaved fragments, simplifying the overall cloning procedure while maintaining directional control
3Ease of manufacture
If DNA manipulation techniques are used to equip fragments with restriction sites, then assembly capability is improved, but alteration of primary nucleotide sequences causes loss of original sequence information
Solution Approach 1:
The invention extracts the restriction site functionality from the traditional prefix/suffix approach and relocates it into the intron sequences. The type IIs restriction sites are embedded within the intron regions, allowing the coding sequences to remain untouched and preserve their original information, while the introns provide the necessary assembly capabilities through their embedded restriction sites
Solution Approach 2:
The intron sequences serve as intermediary elements that mediate between the coding sequences and the cloning process. They contain the type IIs restriction sites needed for assembly but are ultimately removed during splicing, so they facilitate manipulation without permanently altering the final protein-coding sequence information
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 approach allows for efficient and flexible assembly of DNA fragments, enabling the reuse of genetic components in various cloning strategies and facilitating the expression of multiple genes of interest by generating functional fusion polynucleotides that can be transcribed and translated into polypeptides in eukaryotic host cells.
Implementation Method 1
contacting a first and a second polynucleotide with the type IIs restriction endonuclease under conditions that allow for cleavage of the first polynucleotide and the second polynucleotide by said type IIs restriction endonuclease
Implementation Method 2
ligation of the resulting cleavage products, thereby generating the fusion polynucleotide encoding the polypeptide of interest
Implementation Method 3
said fusion polynucleotide, when transcribed in a eukaryotic host cell, is transcribed into a transcript that is processed in said cell
Implementation Method 4
the transcript is processed in said cell so that the intron is removed from said transcript, thereby producing an mRNA encoding the polypeptide of interest
Implementation Method 5
producing an mRNA encoding the polypeptide of interest
Data Source
AI summary
The present invention relates to a method for producing a fusion polynucleotide encoding a polypeptide of interest. The method comprises the steps of providing a first polynucleotide and a second polynucleotide, and contacting said first polynucleotide and second polynucleotide with a type IIs restriction endonuclease and a ligase under conditions that allow for the cleavage of the first polynucleotide and second polynucleotide by said type IIs restriction endonuclease and the ligation of the resulting cleavage products, thereby producing the fusion polynucleotide encoding the polypeptide of interest. The first polynucleotide comprises the 5′ portion of an intron, and the second polynucleotide comprises the 3′ portion of an intron. Further envisaged by the present invention is a polynucleotide encoding a polypeptide of interest, which, when transcribed in a eukaryotic host cell, is transcribed into a transcript comprising at least five introns which are heterologous to said polynucleotide.