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Accurate multiplex gene synthesis from programmable DNA microchips

Nature volume 432, pages 10501054 (23 December 2004) | Download Citation

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Abstract

Testing the many hypotheses from genomics and systems biology experiments demands accurate and cost-effective gene and genome synthesis. Here we describe a microchip-based technology for multiplex gene synthesis. Pools of thousands of ‘construction’ oligonucleotides and tagged complementary ‘selection’ oligonucleotides are synthesized on photo-programmable microfluidic chips1, released, amplified and selected by hybridization to reduce synthesis errors ninefold. A one-step polymerase assembly multiplexing reaction assembles these into multiple genes. This technology enabled us to synthesize all 21 genes that encode the proteins of the Escherichia coli 30S ribosomal subunit, and to optimize their translation efficiency in vitro through alteration of codon bias. This is a significant step towards the synthesis of ribosomes in vitro and should have utility for synthetic biology in general.

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Acknowledgements

DARPA BioComp and DOE GTL provided support for J.T., H.G. and G.C. We thank G. Culver, T. Wu, T. Forster, P. Carr, J. Jacobson and other members of the synthetic biology community for advice; N. Novikov for technical assistance, and E. Nuwaysir and T. Albert for help in designing the Nimblegen arrays. J.T. was supported by a LSRF fellowship. X.Z., E.G. and X.G. thank the NIH, DARPA and the R.A. Welch Foundation for grants.

Author information

Affiliations

  1. Harvard Medical School, 77 Ave Louis Pasteur, Boston, Massachusetts 02115, USA

    • Jingdong Tian
    • , Hui Gong
    •  & George Church
  2. Department of Chemistry, University of Houston, Houston, Texas 77004-5003, USA

    • Nijing Sheng
    •  & Xiaolian Gao
  3. Atactic Technologies Inc., 2575 West Bellfort, Suite 270, Houston, Texas 77054, USA

    • Xiaochuan Zhou
  4. Chemical Engineering Department, University of Michigan, Ann Arbor, Michigan 48109-2136, USA

    • Erdogan Gulari

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Competing interests

X.Z., E.G. and X.G. have potentially competing financial interests in Atactic Technologies. G.C. and J.T. have potentially competing financial interests in SynBio Corp.

Corresponding author

Correspondence to George Church.

Supplementary information

Word documents

  1. 1.

    Supplementary Table 1 and 2

    Table 1: Sequences of the construction oligonucleotides, selection oligonucleotides, oligonucleotide PCR-amplification primers, and gene-end primers; Table 2: Primers for adding His-tags, linkers to construct overlaps, 3 secondary assembly primers, and 1 final PCR assembly flanking primers.

  2. 2.

    Supplementary Methods

    Description of methods for making the synthetic 14,593-nt operon of 21 codon-modified E.coli 30S ribosomal genes and lists of oligonucleotides used.

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DOI

https://doi.org/10.1038/nature03151

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