Template-Based DNA Synthesis for Multi-Site Mutagenesis
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Solution Overview
Problem
Current methods for synthesizing multi-site sequence variants of kilobases in length are inefficient, expensive, and impractical due to size limits, synthesis fidelity issues, and long lead times, particularly for large genomes like those of plants or mammals.
Innovation Solution
The method involves annealing multiple mutagenic primers to a template nucleic acid, followed by contacting the primed template with a polymerase, a ligase, and nucleotide triphosphates to extend and ligate a mutant strand, enabling rapid and scalable synthesis of multi-site sequence variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If de novo DNA synthesis is used to make genetic variants, then complete sequence control is achieved, but time and resources are wasted resynthesizing unchanged portions of the core sequence
Solution Approach 1:
The patent divides the DNA synthesis process into segments: a template strand containing the unchanged core sequence and multiple mutagenic primers containing the desired variants. The primers are annealed to specific regions of the template, allowing selective modification of only the necessary portions while preserving the original sequence elsewhere.
Solution Approach 2:
The patent performs preliminary action by pre-synthesizing the template strand containing the complete core sequence before introducing mutagenic primers. This allows the unchanged portions to be prepared in advance, eliminating the need to resynthesize them during variant generation.
2Manufacturing precision
If de novo DNA synthesis is used to make genetic variants, then complete sequence control is achieved, but cost and labor intensity increase significantly
Solution Approach 1:
The patent segments the synthesis task into a reusable template strand and multiple mutagenic primers. The template strand can be synthesized once and reused for generating numerous variants, dramatically reducing the cost and labor compared to synthesizing each variant completely de novo.
Solution Approach 2:
The patent uses the template strand as a master copy that can be repeatedly copied and modified through annealing with different mutagenic primers. This copying approach allows generation of multiple variants from a single template, reducing overall manufacturing cost and effort.
3Length of moving object
If current DNA synthesis methods are used for kilobase-sized sequences, then synthesis capability is achieved, but size limits and fidelity issues persist
Solution Approach 1:
The patent handles kilobase-sized sequences by segmenting them into a template strand and multiple shorter mutagenic primers. The primers are typically much shorter than the full sequence, allowing high-fidelity synthesis of each primer segment, which are then assembled onto the template with high accuracy.
4Adaptability or versatility
If cellular machinery methods like recombineering or base editing are used, then in vivo modification is achieved, but scalability and automation are limited
Solution Approach 1:
The patent replaces cellular machinery (biological system) with an in vitro chemical system consisting of annealing reactions and polymerase extension. This substitution enables better scalability and automation since the chemical reactions can be performed in standardized laboratory equipment without relying on cellular processes.
5Ease of operation
If commercial PCR mutagenesis kits are used, then simplicity and ease of use are achieved, but only one or two mutations can be made at a time
Solution Approach 1:
The patent merges multiple mutagenic primers into a single reaction mixture, allowing simultaneous introduction of multiple mutations (10s to 100s of variants) in one experiment. This combining approach maintains the simplicity of the protocol while dramatically increasing productivity compared to sequential single-mutation approaches.
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 the efficient generation of 10s to 100s to 1000s of defined sequence variations in kilobases of DNA in vitro, achieving high fidelity and significantly reducing time and cost compared to existing methods.
Implementation Method 1
contacting primed template nucleic acid with a polymerase, a ligase, and nucleotide triphosphates under conditions suitable to extend and ligate a first mutant strand
Implementation Method 2
contacting primed template nucleic acid with a polymerase, a ligase, and nucleotide triphosphates under conditions suitable to extend and ligate a first mutant strand
Implementation Method 3
annealing three or more mutagenic primers to a template nucleic acid, wherein each of the three or more mutagenic primers comprises at least one mutagenic nucleotide in comparison to template strand
Data Source
AI summary
The present disclosure provides to systems, methods, and compositions for template-based DNA, synthesis. Particularly the present disclosure provides systems, methods, and compositions for generating multi-site (e.g., three or more) sequence variants of template nucleic acid strands kilobases (e.g., greater than Ikb) m length.