SBIR-STTR Award

Phase I is the development of a large-array oligonucleotide synthesis instrument that will greatly decrease the cost and simplify construction of mega base-size double-stranded DNA segments. This novel technology will use less than 1/40th of the synthesis reagents, as compared to conventional DNA synthesizers, and will produce oligos in the 100 to 500 nanogram range per reaction. The process enables the synthesis of up to 10,000 overlapping oligos that can be assembled into double-stranded DNA by annealing and ligation. The technology combines conventional oligo nucleotide synthesis chemistry with novel lithographic valving technology to deliver synthesis reagents to a large array of individual reaction chambers. In preliminary data, a prototype of this system was successfully tested. This device uses a series of computer generated valving mats that effect opening or closing of miniaturized oligo synthesis reaction chambers. The prototype allowed synthesis of (100) 32-base oligos at a reagent cost of ~$0.012 per base. These oligos were full-length, DNAs that annealed and ligated to form the predicted 1600 bp fragment of dsDNA coding for an active tet gene. In Phase I, experiments will be carried out to validate the large array oligo synthesis method using an 800-reaction well prototype. Phase I: Aim 1 is to develop a software algorithm that generates graphic dot pattern files that enable the production of silkscreen or laser etched mats to be used for the lithographic valving process. Aim 2 is to generate and test larger scale lithographic mats and determine efficiency of reagent application and cross-contamination. Aim 3 is to synthesize 800 oligos simultaneously using the technology. The resulting overlapping oligos will be annealed and ligated to form a single gene fragment that can be cloned into a plasmid vector and subsequently sequenced to validate the overall process. Aim 4 will be to determine reagent cost of a large-array oligos, and to assess the overall feasibility of the technology as a product or service. In Phase II, the methodology wil be expanded to synthesize up to 10,000 oligos per run. The technology is predicted to generate large-arrays of oligos and hence dsDNA at a cost of <$0.01 per base pair, which is at least ten fold less than current suppliers'prices. The products that result from this work address the rapidly growing field of genome, metabolic pathway, and protein structure manipulation that is important to pharmaceutical drug discovery and development. The market for the product is estimated at $40-50 million annually. Since MacConnell Research already manufactures and sells instruments and kits for molecular biology research, our company can directly market products and services developed by this work after Phase II.

Public Health Relevance:
A novel large-array oligonucleotide synthesizer will be further developed and tested that allows tens of thousands of oligonucleotides to be synthesized simultaneously in 12 hours time at cost of less than 1/40 of current technology. This work opens the door to a significant new era of synthetic biology in which the design, synthesis, and transplantation of large DNA segments can be accomplished allow for the production of many new proteins, drugs, useful bacteria, and other research tools.

Phase II is the development of a multiple-array oligonucleotide synthesis instrument that will greatly decrease the cost and simplify construction of large double-stranded DNA segments. This novel technology uses less than 1/10th of the reagents of conventional DNA synthesizers, and produces 100-200 nanograms of finished oligo per reaction. The process enables the synthesis of up to 10,000 overlapping oligos that can be assembled into double-stranded DNA by annealing and ligation. The methodology combines conventional oligo nucleotide synthesis chemistry with novel lithographic valving technology to deliver synthesis reagents to a large array of individual reaction chambers. In Phase I, a prototype of this system was successfully tested. This device uses a series of computer generated valving mats that effect opening or closing of miniaturized oligo synthesis reaction chambers. The prototype allowed synthesis of (800) 34-base oligos at a reagent cost of ~$0.012 per base. These overlapping oligos were annealed and ligated to form a predicted 13,600 bp fragment of dsDNA coding for active antibiotic resistance genes. The error rate in the resulting synthetic dsDNA was less than one in 1300 bases. In Phase II, the large array oligo synthesis method will be further developed and validated using a 10,000-reaction well prototype. Phase II work includes testing of various thin- film materials for the valving mats, construction and testin of a prototype for simultaneous synthesis of (10,000) overlapping 34-mer oligos in an 80 x 125 array. The resulting oligos will be annealed and ligated to form a set of gene fragments that will be cloned into a plasmid vectors, sequenced, and assayed for activity to validate the overall process. Additional Phase II trials will be carried out to automate the litho mat changing and registration process, as well as reduce the amount of reagents consumed in the synthesis. Synthesized cloned fragments from the above will be assembled into 100 kb or greater dsDNAs using the Gibson assembly. The technology is predicted to generate large-arrays of oligos and dsDNA at a cost of less than $0.01 per base pair. This cost will enable our company offer custom oligo production and gene synthesis at 1/10 that current suppliers' prices. The products that result from this work address the rapidly growing field of genome and metabolic pathway manipulation that is important to drug discovery, as well as the development of industrially useful bacteria. The technology is projected to generate $40 million in annual sales. MacConnell Research can directly market products and services developed by this work after Phase II.

Public Health Relevance Statement:


Public Health Relevance:
A novel large-array oligonucleotide synthesizer will be further developed and tested that allows tens of thousands of oligonucleotides to be synthesized simultaneously in 12 hours' time at cost of less than 1/10 of current technology. This work opens the door to a significant new era of synthetic biology in which the design, synthesis, and transplantation of large DNA segments can be accomplished, allowing for the production of many new proteins, drugs, and useful bacteria.

Project Terms:
Address; Amino Acid Sequence; Antibiotic Resistance; Argon; Bacteria; base; Base Pairing; Biological Assay; Chemicals; Code; computer generated; Containment; cost; Custom; design; Development; Devices; DNA; DNA annealing; drug discovery; ds-DNA; Environment; Escherichia coli; Film; Gases; gene synthesis; Genes; Genome; Hour; Individual; instrument; Lasers; Length; Ligation; Market Research; Marketing; Metabolic Pathway; method development; Methodology; Methods; miniaturize; Molecular Biology; new technology; novel; nucleotide metabolism; Oligonucleotide Microarrays; Oligonucleotides; Peptide Sequence Determination; Pharmaceutical Preparations; Phase; Plant Resins; Plasmid Cloning Vector; plasmid DNA; Plastics; Price; Printing; Process; Production; Proteins; prototype; public health relevance; Reaction; Reagent; receptor; Research; research study; Resistance; resistance gene; Running; Sales; scale up; Series; Services; Silicones; Synthesis Chemistry; synthetic biology; System; Technology; Teflon; Testing; Thick; Time; Transplantation; Work