A Fast Scalable, Automated, One-Pot Process for Assembly of DNA Oligonucleotides into Large Gene Sequences
The production capacity of custom-made DNA sequences is a critical factor in the throughput of synthetic biology workflows, which aim to discover and characterize both novel and existing properties of biomolecules and have wide-ranging applications, from sustainable production of commodity chemicals to gene-based therapies for various illnesses. On-demand DNA production is still expensive and uses specialized equipment that requires significant technical expertise to run, yet current methods are still appreciably error prone. Improvements in the speed, fidelity, and cost of custom nucleic acids are therefore urgently needed. Synthesis of larger DNA constructs that would be commercially available is performed using a sequence of two distinct processes: oligonucleotide synthesis (construction of short DNA sequences that together would make up a finished construct) and oligonucleotide assembly (joining synthesized oligonucleotides into the finished construct). At the current state of the art with respect to both parts of process, downstream oligo assembly throughput currently lags behind that of synthesis; therefore, this project’s focus is on alleviating this bottleneck using a novel assembly method that can selectively amplify, proofread, and assemble subsets of synthesized oligonucleotides from complex mixtures of DNA fragments into their intended finished sequences. Our process uses a unique combination of well-characterized molecular biology methods to develop a novel multiplexable process for the assembly of synthesized DNA oligonucleotides derived from high-throughput microarrays into large-scale sequences with the potential to be more rapid, reliable, automatable, and streamlined than those currently used in industry or described in recently published scientific literature. This process occurs in four sequential stages, each joining multiple smaller fragments (or the previous stage’s outputs) into increasingly larger constructs; while the full process is described in this report, the Phase I effort focused on the first Stage, where the vast majority of the process’s novelty lies. Our results from tests performed using our initially proposed Stage 1 process showed that we can successfully convert a complex mixture of oligonucleotides into functional genes, with the complexity and efficiencies of the assemblies being at least comparable to those described in recent high-profile publications. Optimization experiments for this Stage resulted in increases in the number of DNA fragments that could be accurately joined together at once, as well as the relative amount of off-target sequences that can still be present while creating accurate assemblies, allowing for a single microarray to produce more and larger final constructs using this process. This work demonstrates proof-of-concept of our novel method and a favorable comparison with other recently developed methods; it also presents additional opportunities for optimizing the first and other Stages to further increase the efficiency of all parts of the process and enable the reliable construction of even larger sequences from single microarrays.
- Research Organization:
- Triton Systems, Inc., Chelmsford, MA (United States)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- SC0021603
- OSTI ID:
- 1847301
- Type / Phase:
- SBIR (Phase I)
- Report Number(s):
- DOE-TRITON-21603-3
- Country of Publication:
- United States
- Language:
- English
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