Digital Microfluidic Oligonucleotide Synthesis for Long-Strand Assembly
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Solution Overview
Problem
Current DNA synthesis technologies are limited to producing strands of up to 200 nucleotides in length, making it impossible to efficiently synthesize longer DNA strands required for biotechnology applications, such as gene assembly and hybridization microarrays, due to inefficiencies in nucleotide coupling and the need for template-dependent methods.
Innovation Solution
A method and kit for synthesizing and assembling oligonucleotides in a single device, using a digital microfluidic platform, where oligonucleotides of varying sequences are synthesized, cleaved, and hybridized to form overlapping regions, allowing for the assembly of contiguous strands up to 5000 bases in length through a process involving reversible blocking of nucleotide monomers and enzymatic or chemical joining.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If phosphoramidite technology is used to couple nucleotides one at a time, then DNA synthesis can be performed with high coupling efficiency (99%), but it is mathematically impossible to synthesise DNA longer than 200 nucleotides in acceptable yields
Solution Approach 1:
The patent divides the synthesis process into two distinct stages: (1) synthesis of multiple overlapping oligonucleotide fragments of manageable lengths (e.g., 40-120 nucleotides) using phosphoramidite chemistry, and (2) assembly of these fragments into longer contiguous strands through enzymatic methods. This segmentation allows each fragment to be synthesized with high precision while enabling final products to exceed 200 nucleotides in length.
Solution Approach 2:
The patent introduces enzymatic processes as intermediary steps between fragment synthesis and final assembly. Specifically, DNA polymerases and ligases are used as mediators to extend and join the synthesized oligonucleotide fragments, bridging the gap between chemically synthesized segments and the desired long contiguous strand.
2Productivity
If template-dependent DNA polymerases are used for sequencing-by-synthesis, then strands of between 500-1000 bps long can be produced, but the technology is not suitable for de novo nucleic acid synthesis because of the requirement for an existing nucleic acid strand to act as a template
Solution Approach 1:
The patent segments the de novo synthesis task into synthesizing multiple short oligonucleotide fragments without templates, then assembling them using template-dependent polymerases. Each fragment can be synthesized independently (e.g., by phosphoramidite chemistry or other template-independent methods), and the assembly phase leverages the high elongation capability of template-dependent polymerases to achieve long final strands.
Solution Approach 2:
The patent performs preliminary synthesis of multiple overlapping oligonucleotide fragments before the assembly step. These fragments are prepared in advance with designed overlaps that will serve as templates during the subsequent polymerase-mediated extension and joining process, enabling de novo synthesis of long strands.
3Ease of operation
If TdT is used for controlled de novo single-stranded DNA synthesis, then the need for an anhydrous environment is removed and various polymers incompatible with organic solvents can be used, but TdT has not been shown to efficiently add nucleoside triphosphates containing 3′-O-reversibly terminating moieties for building up a nascent single-stranded DNA chain
Solution Approach 1:
The patent uses TdT for the preliminary synthesis of oligonucleotide fragments under relaxed environmental conditions (aqueous environment, compatible with various polymers), then switches to template-dependent polymerases for the assembly phase where high efficiency in adding nucleotides is required. This segmentation allows TdT's environmental advantages to be utilized without compromising overall synthesis efficiency.
Solution Approach 2:
The patent introduces template-dependent DNA polymerases as intermediary enzymes to bridge the gap between TdT-synthesized fragments and the final long strand. These polymerases efficiently add nucleoside triphosphates (including those with 3′-O-reversibly terminating moieties) during the assembly phase, compensating for TdT's limitations while preserving the environmental benefits gained in the fragment synthesis phase.
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 efficient production of long oligonucleotide strands, overcoming the limitations of existing methods by allowing strands longer than 200 nucleotides to be synthesized and assembled, facilitating applications in biotechnology and pharmaceuticals.
Implementation Method 1
using a terminal deoxynucleotidyl transferase to add a nucleoside triphosphate to a 3'-end of the immobilised oligonucleotide sequences
Implementation Method 2
wherein the nucleoside triphosphates are reversibly terminating nucleoside triphosphates
Implementation Method 3
hybridizing at least two of the cleaved oligonucleotides to each other, to form a splint, and hybridizing one end of the splint to one of the immobilized oligonucleotide sequences
Implementation Method 4
joining at least one of the cleaved oligonucleotides to the immobilised oligonucleotide sequences, thereby preparing a contiguous oligonucleotide sequence
Data Source
AI summary
The invention relates to methods and kits for the synthesis and assembly of oligonucleotides into contiguous strands. The oligonucleotides can be synthesised and assembled in the same device, allowing production of strands longer than can be prepared using base by base synthesis alone.