Hierarchical DNA Assembly from a Universal Oligonucleotide Library
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Solution Overview
Problem
Existing methods for synthesizing oligonucleotides suffer from limitations in sequence fidelity and length, leading to errors and inefficiencies in producing oligonucleotides of desired sequences.
Innovation Solution
A method involving the use of a library of overlapping oligonucleotides, annealing, ligation, and amplification steps to synthesize DNA molecules of any desired sequence with high fidelity, using a universal library of fewer than 10,000 oligonucleotides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If chemical synthesis via solid-phase sequential coupling is used, then oligonucleotides can be produced automatically, but side reactions and base errors occur limiting the length and fidelity
Solution Approach 1:
The patent divides the oligonucleotide synthesis process into multiple sequential stages: initial chemical synthesis of short oligonucleotides (1-200 nt), enzymatic extension to intermediate lengths (200-1000 nt), and final assembly of full-length products (1000-10000+ nt). Each stage uses optimized methods appropriate for that size range, thereby maintaining high fidelity throughout the overall process while achieving both automation and length extension.
Solution Approach 2:
The patent introduces enzymatic processes as intermediary steps between chemical synthesis and final product assembly. Template-directed polymerases and ligases serve as mediators that extend and join oligonucleotide fragments with high fidelity, bridging the gap between the automated chemical synthesis stage and the final high-fidelity product, thereby resolving the contradiction between automation and precision.
2Length of moving object
If enzymatic methods using terminal deoxynucleotidyl transferase are used, then oligonucleotide extension can be achieved, but the enzyme shows strong bias for specific nucleotide bases and does not reliably add nucleotides in the desired order
Solution Approach 1:
The patent uses template-directed polymerases as intermediaries that read a template strand and incorporate nucleotides complementary to the template in the desired order. This template-directed mechanism eliminates the base bias problem of TdT while maintaining the ability to extend oligonucleotides to desired lengths with high sequence fidelity.
Solution Approach 2:
The patent changes the fundamental parameter of how nucleotide incorporation is directed: instead of using template-independent TdT that adds nucleotides based on enzyme bias, the system uses template-directed polymerization where the template sequence determines the incorporation order, thereby achieving both length extension and sequence accuracy.
3Productivity
If chemical synthesis methods are used, then oligonucleotides can be produced, but the length of oligonucleotides produced is limited due to side reactions and base errors
Solution Approach 1:
The patent segments the synthesis process into distinct phases: chemical synthesis produces short high-fidelity fragments (1-200 nt), which are then enzymatically extended to intermediate lengths (200-1000 nt), and finally assembled into full-length products (1000-10000+ nt). This segmentation allows each stage to operate within its optimal performance range, thereby achieving both productivity and length extension.
Solution Approach 2:
The patent performs preliminary chemical synthesis of short oligonucleotide fragments with high fidelity before using enzymatic methods to extend them to full length. This preliminary action creates a foundation of accurate short sequences that can be reliably extended, thereby overcoming the length limitation of direct chemical synthesis while maintaining overall sequence fidelity.
4Adaptability or versatility
If a universal library of oligonucleotides is used, then any possible DNA sequence can be synthesized, but the library size would typically be very large
Solution Approach 1:
The patent segments the universal library into modular components: a core set of short oligonucleotide fragments (1-200 nt) that can be chemically synthesized, which serve as building blocks for enzymatic extension and assembly. This segmentation allows a relatively small library of core fragments to generate a vast diversity of full-length sequences through combinatorial assembly, thereby achieving universality with reduced library size.
Solution Approach 2:
The patent designs the oligonucleotide library with universal features: standardized overhang sequences, common adapter regions, and modular structures that allow any fragment to potentially combine with any other fragment. This universality enables a small library of multi-functional building blocks to generate any possible DNA sequence through systematic assembly, reducing the total number of oligonucleotides needed while maintaining comprehensive coverage.
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
Enables the synthesis of DNA molecules with an error rate of less than 1 error per 2,000 nucleotides and lengths up to 10,000 bp, facilitating the assembly of any possible DNA sequence.
Implementation Method 1
annealing at least two oligonucleotides to at least two anchor strands
Implementation Method 2
annealing at least two oligonucleotides comprising a variable sequence to at least two anchor strands so that the at least two oligonucleotides abut one another at their variable sequences
Implementation Method 3
ligating the at least two oligonucleotides annealed to the at least two anchor strands at the variable sequences of their 5′ and 3′ ends
Implementation Method 4
performing a step of selectively digesting the anchor strands to produce a circular DNA molecule
Implementation Method 5
performing an amplification step on the first single-stranded circular DNA molecule to produce a first double-stranded DNA molecule
Data Source
AI summary
The invention provides methods for synthesizing a product DNA molecule of any possible DNA sequence from a universal library of overlapping oligonucleotides. The method involves combining a plurality of the overlapping oligonucleotides in a reaction pool, where the sequences of the plurality of oligonucleotides comprise at least a sub-sequence of the product DNA molecule. The method also involves annealing the plurality of oligonucleotides, performing a ligation step, a selective digestion step, and an amplification step to thereby synthesize a sub-sequence of the product DNA molecule, and a produce DNA molecule using hierarchical assembly. The invention can be used to synthesize a DNA molecule of any possible sequence from the universal library, which can be accomplished through a hierarchal assembly scheme. In one embodiment the universal library comprises fewer than 10,000 pre-manufactured oligonucleotides that can be synthesized into the any possible DNA sequence. The product DNA molecule can be more than 150 base pairs long.