The ability to understand genome biology and the consequences of genetic variation on individual health depends heavily on technologies that allow researchers to read and write DNA. Sequencing technologies have recently seen dramatic improvements that have made personal genomes affordable for virtually any laboratory and even directly available to consumers. Yet the corresponding DNA synthesis technologies lag far behind these developments, causing a major hindrance in synthetic biology efforts to study genes, variants, and genomes of interest by synthesizing them. This proposal aims to develop a novel DNA synthesis technology to address the greatest challenge faced by current platforms: maintaining sufficient accuracy for precision applications and throughput for large-scale applications while remaining cost-effective for accessibility. To achieve this, the traditional phosphoramidite method of synthesizing DNA oligonucleotides will be adapted onto nanoparticular beads, which will be moved through droplets containing synthesis reagents along a plasmonic surface array. This `conveyer belt' will be optically controlled via C-shaped engravings (CSEs) that concentrate light from below, serving as optical traps. In this way, the beads and reagent droplets can be individually, rapidly transported to specific optical traps in multiple lanes simply by changing the illumination wavelengths, allowing millions of unique oligonucleotides to be synthesized simultaneously on a single array. By tailoring the reagent droplet size and concentration depending on synthesis scale, the method will be optimized to target the entire bead surface, maximize yield, and eliminate excess reagent usage. Quality will be assessed by testing synthesis of diverse DNA sequences. The main advantages of this novel DNA synthesis platform will include: 1) faster reactions (cycle time 45 sec), 2) lower error rate due to decreased acid exposure (<1:1000), 3) high yield (>5 attomoles/bead), 4) increased length of oligonucleotides (>300 bases) due to cleaner synthesis, 6) significantly less hazardous waste production, 7) generation of >25 million unique oligonucleotide sequences in a single run that can be individually isolated for downstream applications, and 8) a cost of $0.0000001/base, two orders of magnitude less than the least expensive method currently available. A DNA synthesis technology with these properties will enable unprecedented genomic investigations, allowing researchers to test the functional and clinical impact of thousands of genes and genetic variants.
DNA sequencing technology has progressed at a tremendous rate, yet our ability to write DNA is far behind our ability to read it. Synthesis technologies have been an extremely valuable tool for studying the function of genes and genomes, but no current technologies address all of the most important needs for throughput, accuracy, and cost-effectiveness. Here we propose to develop a novel DNA synthesis platform that builds DNA strands on beads that are precisely moved along a nano-optical `conveyer belt,' allowing for unparalleled speed and throughput as well as vastly reduced error rates and costs.
