Nucleic Acid Assembly via Polymerase Extension and Hybridization
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Solution Overview
Problem
Current gene synthesis methods lack efficiency in high throughput synthesis and assembly of larger nucleic acid molecules, often requiring labor-intensive and costly processes, and struggle with achieving high sequence fidelity and miniaturization.
Innovation Solution
The development of methods and compositions for high throughput synthesis and assembly of nucleic acid molecules, involving the sorting and pooling of oligonucleotides, assembly of double-stranded target nucleic acids using polymerase-mediated extension, and error correction steps, utilizing specific polymerases and kits to achieve predefined sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional gene synthesis methods are used, then sequence fidelity can be maintained, but productivity and throughput are low
Solution Approach 1:
The patent divides the gene synthesis process into modular assembly steps where oligonucleotides are synthesized separately and then assembled into larger nucleic acid molecules through controlled hybridization and polymerase extension. This segmentation allows parallel processing of multiple oligonucleotides while maintaining sequence fidelity through individual verification and controlled assembly conditions.
Solution Approach 2:
The patent incorporates error correction mechanisms where the assembled nucleic acid molecules undergo verification steps including sequencing and comparison against the intended design sequence. Errors are identified and corrected through feedback loops that involve re-synthesis or selection of correct sequences, thereby maintaining high sequence fidelity despite high-throughput parallel processing.
2Ease of manufacture
If chemically synthesized oligonucleotides are assembled traditionally, then sequence accuracy can be verified, but the process is labor-intensive and costly
Solution Approach 1:
The patent combines multiple assembly operations into a single reaction vessel where multiple oligonucleotides hybridize and extend simultaneously. This merging of parallel operations into unified assembly reactions eliminates the need for separate manual handling steps, reducing labor intensity and cost while maintaining high throughput through the simultaneous processing of multiple nucleic acid assemblies.
Solution Approach 2:
The patent employs self-assembly mechanisms where oligonucleotides automatically hybridize to their complementary sequences and undergo polymerase-mediated extension without extensive manual intervention. The system uses inherent molecular recognition and enzymatic processes to perform the assembly work, minimizing labor requirements while maximizing productivity through autonomous molecular self-organization.
3Productivity
If assembly methods are simplified for high throughput, then productivity increases, but miniaturization and parallelization become difficult
Solution Approach 1:
The patent transitions from traditional planar assembly approaches to three-dimensional microfluidic reaction environments where oligonucleotides are assembled in suspended droplets or within micro-wells. This dimensional change enables miniaturization of reaction volumes while maintaining high throughput through parallel processing of numerous micro-reactions simultaneously, thereby achieving both high productivity and adaptability to scaled-down formats.
Solution Approach 2:
The patent develops universal assembly protocols and reagent systems that function effectively across multiple scales from microliter to nanoliter volumes. The same hybridization and polymerase extension mechanisms work whether processing a single large reaction or thousands of miniaturized parallel reactions, providing versatility for both high-throughput production and miniaturized applications without requiring separate optimized protocols.
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 enables efficient, cost-effective, and high-fidelity synthesis of larger nucleic acid molecules, facilitating miniaturization and parallelization of gene synthesis workflows, thereby reducing costs and improving sequence accuracy.
Implementation Method 1
hybridization of the population of single-stranded oligonucleotides to one another and to the single-stranded termini of the polynucleotide in a pre-determined order
Implementation Method 2
polymerase-mediated extension of the free 3' ends of the hybridized oligonucleotides and the polynucleotide strands to generate a double-stranded non-covalently closed circularized assembly product
Implementation Method 3
at least partial denaturation of the linear double-stranded polynucleotide to obtain single-stranded termini
Data Source
Figure 1
Figure 2(a)~2(d)
Figure 3
AI summary
The present disclosure generally relates to compositions, methods and kits for sorting and assembly of nucleic acid molecules, such as synthetic single-stranded oligonucleotides into larger nucleic acid molecules by mixing said single-stranded oligonucleotides with a linear double-stranded vector. The disclosed compositions and methods allow for miniaturization, parallelization, high throughput production and cost reduction of nucleic acid assembly and gene synthesis.