High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis (original) (raw)

Nature Biotechnology volume 27, pages 1173–1175 (2009)Cite this article

Abstract

We present a method that harnesses massively parallel DNA synthesis and sequencing for the high-throughput functional analysis of regulatory sequences at single-nucleotide resolution. As a proof of concept, we quantitatively assayed the effects of all possible single-nucleotide mutations for three bacteriophage promoters and three mammalian core promoters in a single experiment per promoter. The method may also serve as a rapid screening tool for regulatory element engineering in synthetic biology.

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Acknowledgements

The authors would like to thank M.G. Surette (Univ. of Calgary) for the generous gift of the pCS26 plasmid used for the luciferase assays; E. LeProust and W. Woo (Agilent Technologies) for array-derived oligonucleotides libraries and E. Turner, J.B. Hiatt, S. Ng, J. Kitzman, R. Monnat, B. Stone, A. Dudley and N. Goddard for helpful discussions. D.P. holds a Career Award at the Scientific Interface from the Burroughs Wellcome Fund.

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Authors and Affiliations

  1. Department of Genome Sciences, University of Washington, Seattle, Washington, USA
    Rupali P Patwardhan, Choli Lee, David L Young & Jay Shendure
  2. Department of Biological Sciences, Columbia University, New York, New York, USA
    Oren Litvin & Dana Pe'er
  3. Center for Computational Biology and Bioinformatics, Columbia University, New York, New York, USA
    Oren Litvin & Dana Pe'er

Authors

  1. Rupali P Patwardhan
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  2. Choli Lee
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  3. Oren Litvin
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  4. David L Young
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  5. Dana Pe'er
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  6. Jay Shendure
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Contributions

The project was conceived and experiments planned by R.P.P., C.L., D.P. and J.S. Experiments were performed by R.P.P., C.L. and D.L.Y. Data analysis was performed by R.P.P. and O.L. The manuscript was written by R.P.P. and J.S. All aspects of the study were supervised by J.S.

Corresponding authors

Correspondence toRupali P Patwardhan or Jay Shendure.

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Patwardhan, R., Lee, C., Litvin, O. et al. High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis.Nat Biotechnol 27, 1173–1175 (2009). https://doi.org/10.1038/nbt.1589

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