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

• Published in: Nature biotechnology Vol. 28; no. 12; pp. 1295 - 1299
• Main Authors: Kosuri, Sriram, Eroshenko, Nikolai, LeProust, Emily M, Super, Michael, Way, Jeffrey, Li, Jin Billy, Church, George M
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01.12.2010; Nature Publishing Group
• Subjects: 631/1647/1888/1890; 631/1647/2017/2079; 631/92/95; Agriculture; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cloning, Molecular - methods; Deoxyribonucleic acid; Diverse techniques; DNA; DNA - chemical synthesis; DNA - chemistry; DNA microarrays; DNA synthesis; Fundamental and applied biological sciences. Psychology; Genes, Synthetic; Genetic engineering; Genetic technics; Humans; letter; Life Sciences; Methods; Methods. Procedures. Technologies; Molecular and cellular biology; Nucleic Acid Amplification Techniques - methods; Nucleic Acid Hybridization - methods; Oligonucleotide Array Sequence Analysis - instrumentation; Oligonucleotide Array Sequence Analysis - methods; Oligonucleotides; Physiological aspects; Process engineering; Protein synthesis; Semiconductors; Synthetic Biology - methods; Synthetic digonucleotides and genes. Sequencing; United States; Amplification; Synthesis; Gene; DNA; DNA chip; Joining; System on a chip; Selectivity; Method
• ISSN: 1087-0156; 1546-1696; 1546-1696
• DOI: 10.1038/nbt.1716

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 biotechnology 1 . Currently, the reliance on column-synthesized oligonucleotides as a source of DNA limits further cost reductions in gene synthesis 2 . Oligonucleotides from DNA microchips can reduce costs by at least an order of magnitude 3 , 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.