Tailored Multi-Site Combinatorial Assembly for Gene Variant Generation
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Solution Overview
Problem
Current methods for generating specific gene variants and combinatorial gene libraries are laborious and inefficient, particularly when attempting to introduce multiple mutations at various sites, often resulting in excess mutations or requiring complex multi-step processes.
Innovation Solution
A tailored multi-site combinatorial assembly method involving the use of multiple primers in a single reaction mixture, where at least three or two primers with distinct mutations are added to a double-stranded template, followed by a polymerase extension reaction, and subsequent transformation into cells without the need for ligation, allowing for the recovery and selection of modified polynucleotides with specific mutations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If multiple oligonucleotides are used simultaneously to generate mutations at multiple sites, then the ability to create combinatorial gene libraries is improved, but the level of incorporation of each primer increases to more than 75%, resulting in excess mutations in single DNA molecules
Solution Approach 1:
The method segments the mutagenesis process into distinct phases: first annealing oligonucleotides to the template DNA, then using a flap endonuclease to selectively process the annealed oligos. This segmentation allows control over primer incorporation by preventing simultaneous incorporation of multiple oligonucleotides, thereby avoiding excess mutations while still enabling combinatorial library generation.
Solution Approach 2:
The patent introduces a flap endonuclease as an intermediary enzyme that processes the annealed oligonucleotides. This intermediary selectively removes excess or incorrectly incorporated primers, ensuring that only the desired single mutation per DNA molecule is retained. The flap endonuclease acts as a mediator between the multiple oligonucleotides and the template DNA, controlling the incorporation process to prevent excess mutations.
2Manufacturing precision
If conventional PCR-based ligation/recombination methods are used to introduce multiple mutations, then gene modifications can be achieved, but the process becomes laborious and requires multiple steps
Solution Approach 1:
The method merges multiple functions into a single reaction vessel: annealing of multiple oligonucleotides to the template DNA, flap endonuclease processing to control primer incorporation, and generation of combinatorial mutants all occur simultaneously in one pot. This eliminates the need for separate PCR amplification, ligation, and recombination steps required by conventional methods, significantly reducing process complexity while maintaining gene modification capability.
Solution Approach 2:
The single reaction system performs multiple functions: it serves as an annealing reaction, a flap endonuclease processing reaction, and a mutagenesis reaction all at once. The same reaction mixture generates the combinatorial library without requiring separate steps for each function, making the process universally applicable to multiple mutation scenarios without increasing complexity.
3Manufacturing precision
If error-prone PCR or site-directed mutagenesis kits are used, then single site mutations can be generated, but making modifications at multiple regions becomes laborious
Solution Approach 1:
The method segments the target gene into multiple regions, each targeted by a specific oligonucleotide with a desired mutation. By designing multiple oligos that anneal to different regions simultaneously and using flap endonuclease to process them, the system maintains single-site mutation accuracy for each region while efficiently generating all possible combinations across multiple regions in a single reaction, greatly improving productivity.
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 and rapid generation of gene variants with multiple mutations at multiple sites, simplifying the process and reducing the complexity of generating combinatorial libraries, while minimizing the risk of excess mutations, and allowing for tailored library creation based on specific needs.
Implementation Method 1
subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers
Implementation Method 2
adding at least three primers to a double stranded template polynucleotide in a single reaction mixture
Data Source
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AI summary
The present invention provides a novel method of producing a plurality of modified polynucleotides having different combinations of various mutations at multiple sites by a tailored multi-site combinatorial assembly, comprising adding at least two or at least three primers to a double stranded template polynucleotide in a single reaction mixture, wherein the primers are not overlapping, and wherein each of the primers comprise at least one mutation different from the other primers, wherein at least one primer is a forward primer that can anneal to a minus strand of the template and at least one primer is a reverse primer that can anneal to a plus strand of the template, and subjecting the reaction mixture to a polymerase extension reaction to yield a plurality of extended modified polynucleotides from the at least three primers. The method can be performed without employing a ligation step prior to transforming the extended modified polynucleotides into a cell. The plurality of extended modified polynucleotides can be treated with an enzyme for destroying the template polynucleotide prior to transforming in to the cell.