Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips

Abstract

Long DNA molecules, such as those encoding genes, can be assembled from short oligonucleotides created on a microarray. Kosuri et al. improve the fidelity and scalability of this process, enabling synthesis of 40 antibody fragments having repetitive regions and other challenging sequence features. Development of cheap, high-throughput and reliable gene synthesis methods will broadly stimulate progress in biology and biotechnology1. Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis2. Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude3,4,5, yet efforts to scale their use have been largely unsuccessful owing to the high error rates and complexity of the oligonucleotide mixtures. Here we use high-fidelity DNA microchips, selective oligonucleotide pool amplification, optimized gene assembly protocols and enzymatic error correction to develop a method for highly parallel gene synthesis. We tested our approach by assembling 47 genes, including 42 challenging therapeutic antibody sequences, encoding a total of ∼35 kilobase pairs of DNA. These assemblies were performed from a complex background containing 13,000 oligonucleotides encoding ∼2.5 megabases of DNA, which is at least 50 times larger than in previously published attempts.