ABSTRACT In recent years the need for long custom synthetic DNA has rapidly increased. While chemical synthesis of oligonucleotides of up to 100 base pairs is a relatively simple and established process, chemical synthesis of longer oligonucleotides is inefficient and costly, resulting in a product with high accumulated errors and limited yield. Current approaches to making long DNAs instead generate shorter overlapping DNA oligos and biochemically assemble them to create the desired DNA sequence. In addition to being confounded by issues of secondary structure and repetitive sequences, these methods are laborious, time-consuming and relatively expensive. This project proposes the development of a DNA synthesizer capable of manufacturing DNA oligonucleotides of over two-thousand base pairs. The synthesizer will be a semiconductor-based device, about the size of a USB thumb-drive, and will employ enzyme-catalyzed DNA synthesis. After loading the device with a few microliters of reagent, DNA will be synthesized at the single molecule level in a nano reactor cell (NRC). A high-fidelity DNA amplification will be used to generate larger quantities of DNA post synthesis. Iridia has developed a novel biochemistry for the DNA synthesis based on an engineered topoisomerase, and have shown this biochemistry to be compatible with solid-state nanopores in a semiconductor chip. The key innovation concepts are: (1) the reagents in the NRC are segregated by nanopores, such that only DNA (and not enzymes) can move through the nanopores, allowing electrophoretic control of the sequential reactions; (2) the NRC enzyme loading process, wherein activated enzymes charged with DNA bases are introduced through the appropriate microfluidic channels; and (3) engineering of a secondary DNA structure which can be observed via monitoring of the nanopore current, enabling real time quality control of the synthesis reaction. The first phase of this project will focus on optimizing and characterizing the biochemistry of the platform. The second phase will be aimed to integrate the biochemistry into a semiconductor-based nanopore device to enable single molecule DNA synthesis with real-time monitoring. A high-fidelity amplification approach will then be used to generate larger quantities of DNA for the user. Iridia, Inc. expect this approach to enable synthesis of DNA of several thousand nucleotides with a very low error rate.