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

Nature Biotechnology volume 28, pages 1295–1299 (2010)Cite this article

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Abstract

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.

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Figure 1: Schematic for scalable gene synthesis from OLS pool 2.
Figure 2: Gene synthesis products.
Figure 3: Characterization of products from OLS pools 1 and 2.

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Acknowledgements

This work was supported by the US Office of Naval Research (N000141010144), National Human Genome Research Institute Center for Excellence in Genomics Science (P50 HG003170), Department of Energy Genomes to Life (DE-FG02-02ER63445), Defense Advanced Research Projects Agency (W911NF-08-1-0254) and the Wyss Institute for Biologically Inspired Engineering (all to G.M.C.). We thank H. Padgett for providing ErrASE and expertise during optimization and J. Boeke for advice on gene assembly protocols. We also thank S. Raman, F. Vigneault and F. Zhang for critical readings of the manuscript, G. Dantas for pZE21 (Washington University), F. Isaacs (Yale University) for pZE21G and J.S. Workman (Wyss Institute) for pSecTag2A.

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Author notes

  1. Jin Billy Li

    Present address: Present address: Department of Genetics, Stanford University, Stanford, California, USA.,

  2. Sriram Kosuri and Nikolai Eroshenko: These authors contributed equally to this work.

Authors and Affiliations

  1. Wyss Institute for Biologically Inspired Engineering, Boston, Massachusetts, USA

    Sriram Kosuri, Nikolai Eroshenko, Michael Super, Jeffrey Way & George M Church

  2. Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA

    Sriram Kosuri, Jin Billy Li & George M Church

  3. Harvard School of Engineering and Applied Sciences, Cambridge, Massachusetts, USA

    Nikolai Eroshenko

  4. Agilent Technologies, Santa Clara, California, USA

    Emily M LeProust

Authors
  1. Sriram Kosuri
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  3. Emily M LeProust
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  4. Michael Super
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  6. Jin Billy Li
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  7. George M Church
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Contributions

S.K. and N.E. wrote the paper with contributions from all authors; S.K. and G.M.C. conceived the study; S.K. wrote all algorithms and designed all sequences; S.K. and N.E. designed and performed all experiments; E.L. provided the oligonucleotides libraries; M.S. and J.F. designed the single-chained versions of commercial antibodies; J.B.L. performed the OLS high-throughput sequencing experiment and provided critical advice on the processing of subpools.

Corresponding authors

Correspondence to Sriram Kosuri or Nikolai Eroshenko.

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Competing interests

E.M.L. is an employee of Agilent Technologies, the commercial provider of OLS pools. G.M.C. is a co-founder of an early-stage startup company involved in gene synthesis. S.K., N.E. and G.M.C. are named inventors on a patent application on technologies described in this article. S.K. is a post-doctoral fellow whose future employment prospects depend upon refereed publications.

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Supplementary Tables 1–3, Supplementary Figs. 1–11, Supplementary Notes 1, 2 and Supplementary Methods (PDF 4391 kb)

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Kosuri, S., Eroshenko, N., LeProust, E. et al. Scalable gene synthesis by selective amplification of DNA pools from high-fidelity microchips. Nat Biotechnol 28, 1295–1299 (2010). https://doi.org/10.1038/nbt.1716

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