This Small Business Innovation Research (SBIR) Phase I project attempts to radically reduce the cost of error-free oligonucleotides for use with gene synthesis. DNA Synthesis of large molecules is done by the assembly of many short oligonucleotide fragments of DNA 60-100bp in length. Currently, each DNA fragment is synthesized in relatively small numbers at an excessive macroscopic scale that incurs a large manufacturing overhead in its production costs. This sets a high cost floor for the entire DNA synthesis process. One route to making cheaper oligonucleotides is to synthesize them in large sets on microarrays. However, array-synthesized DNA is both extremely error-prone and produced as dilute, complex mixtures. This proposed project will use massively parallel sequencers to sequence clonally amplified copies of DNA species sampled from the microarray in order to sort every species apart from one another as well as to identify correctly synthesized oligonucleotides from incorrect ones. Further, it is proposed to use focused laser pulses in a custom laser ejection device to eject and recover desired subsets of perfect oligos from micron-scale sequenced colonies into multiwell plates for assembly into genes. The goal is to be able to recover tens of thousands of sequence-verified oligonucleotides in several hours from sequencer flowcells. The broader impact/commercial potential of this project is to achieve truly disruptive cost decreases in the DNA synthesis of arbitrary genetic information. With the abundance of sequencing data, it is possible to imagine entering a new era of "constructive" biology, where in addition to classical reductive experiments on components, it will be possible to test our understanding of genetic- and protein-based circuits by synthesizing new designs and measuring their discrepancy from predicted behaviors. The low-cost industrial production of arbitrary synthetic DNA has the potential to change the practice of biology such that it becomes cost effective to engineer whole genetic pathways and even genomes, accelerating the development of bioengineering, synthetic biomaterials production, as well as medical and research applications
