Oligonucleotide Library Ligation for Contiguous Randomised Codons
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Solution Overview
Problem
Existing methods for producing oligonucleotide libraries with contiguous randomised codons are limited, as they require the production of randomised template oligonucleotides, which complicates the randomisation of three or more consecutive codons and results in low yields and high unwanted sequence percentages.
Innovation Solution
A method involving the ligation of randomisation oligonucleotides with blunt ends to starter oligonucleotides, using DNA ligase, followed by PCR amplification and purification, allows for the production of oligonucleotide libraries with multiple contiguous randomised codons without the need for randomised template oligonucleotides, reducing unwanted sequences and increasing yield.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If randomised template oligonucleotides are used for producing oligonucleotide libraries with contiguous randomised codons, then the randomisation of three or more consecutive codons can be achieved, but the process becomes complex and yields low results with high unwanted sequence percentages
Solution Approach 1:
The method divides the randomisation process into separate steps: first synthesising non-randomised template oligonucleotides with specific sequences, then using randomisation oligonucleotides that can be ligated to create contiguous randomised codons. This segmentation allows complex randomisation patterns to be built systematically without requiring complex template structures.
Solution Approach 2:
The patent performs preliminary synthesis of non-randomised template oligonucleotides with specific sequences before introducing randomisation. By preparing the template structure in advance with defined sequences, the subsequent randomisation step can focus only on introducing variability at specific positions, simplifying the overall process.
2Adaptability or versatility
If randomised template oligonucleotides are produced prior to using selection oligonucleotides, then contiguous randomised codons can be generated, but the mass and complexity of the template oligonucleotide limits the number of codons that can be randomised
Solution Approach 1:
The method segments the template structure into smaller, manageable non-randomised oligonucleotide units that can be ligated with randomisation oligonucleotides. This segmentation allows multiple randomised codons to be introduced without requiring a single massive template oligonucleotide, thereby increasing the number of randomisable codons.
Solution Approach 2:
The patent transitions from a single-strand template approach to a double-strand approach where non-randomised template oligonucleotides are ligated with randomisation oligonucleotides. This dimensional change allows for more flexible and extensive randomisation without being constrained by the mass limitations of single-strand templates.
3Quantity of substance
If traditional hybridisation methods are used for producing DNA libraries, then conventional screening can be performed, but only small quantities of DNA constructs are produced making manipulation difficult
Solution Approach 1:
The patent replaces the mechanical hybridisation process with a ligation-based approach using DNA ligase to join non-randomised template oligonucleotides with randomisation oligonucleotides. This substitution enables the production of larger quantities of DNA constructs with defined sequences, improving both quantity and ease of manipulation.
Solution Approach 2:
The method changes the fundamental parameter of DNA construction from hybridisation-based assembly to ligation-based assembly. This parameter change allows for the production of larger quantities of DNA constructs with specific sequences, making them easier to manipulate and screen compared to traditional hybridisation methods.
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 efficient production of oligonucleotide libraries with 1 to 10 contiguous randomised codons, improving yield and reducing non-MAX codons, facilitating the creation of peptide libraries for screening and expression.
Implementation Method 1
The technique now identified and described by the inventors unexpectedly allows the production of contiguous randomised codons without the need to produce randomised template oligonucleotides
Implementation Method 2
followed by PCR amplification and purification, allows for the production of oligonucleotide libraries with multiple contiguous randomised codons
Implementation Method 3
the predefined oligonucleotide comprises a nucleotide sequence coding for a restriction endonuclease recognition site capable of being recognised by a restriction endonuclease, the restriction endonuclease capable of cleaving the predefined oligonucleotide upstream or downstream of the endonuclease recognition at a predetermined cleavage site to create a blunt ended cut
Data Source
Figure 1A~1C
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AI summary
The invention relates to a method of producing an oligonucleotide library comprising a plurality of oligonucleotides, each oligonucleotide in the library having at least one predetermined position, a randomisation codon selected from a defined group of codons, the codons within said defined group coding for different amino acids. Vector, host cells containing such libraries and kits for the production of such libraries are also provided.