Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

High-resolution analysis of DNA regulatory elements by synthetic saturation mutagenesis

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Synthetic saturation mutagenesis of a bacteriophage promoter.
Figure 2: Mutagenesis of mammalian core promoters.

References

  1. Wang, X., Xuan, Z., Zhao, X., Li, Y. & Zhang, M . Genome Res. 19, 266–275 (2009).

    Article  CAS  Google Scholar 

  2. Jin, V.X., Singer, G.A., Agosto-Perez, F.J., Liyanarachchi, S. & Davuluri, R.V . BMC Bioinformatics 7, 114 (2006).

    Article  Google Scholar 

  3. Abeel, T., Saeys, Y., Bonnet, E., Rouzé, P. & Van de Peer, Y . Genome Res. 18, 310–323 (2008).

    Article  CAS  Google Scholar 

  4. Johnson, D.S., Mortazavi, A., Myers, R.M. & Wold, B . Science 316, 1497–1502 (2007).

    Article  CAS  Google Scholar 

  5. de Hoon, M. & Hayashizaki, Y . Biotechniques 44, 627–628, 630, 632 (2008).

    Article  CAS  Google Scholar 

  6. Carninci, P. et al. Nat. Genet. 38, 626–635 (2006).

    Article  CAS  Google Scholar 

  7. Baliga, N.S . Biol. Proced. Online 3, 64–69 (2001).

    Article  CAS  Google Scholar 

  8. Kinkhabwala, A. & Guet, C.C . PLoS ONE 3, e2030 (2008).

    Article  Google Scholar 

  9. Gertz, J., Siggia, E. & Cohen, B . Nature 457, 215–218 (2008).

    Article  Google Scholar 

  10. Cleary, M.A. et al. Nat. Methods 1, 241–248 (2004).

    Article  CAS  Google Scholar 

  11. Shin, I., Kim, J., Cantor, C.R. & Kang, C . Proc. Natl. Acad. Sci. USA 97, 3890–3895 (2000).

    Article  CAS  Google Scholar 

  12. Goh, E.B. et al. Proc. Natl. Acad. Sci. USA 99, 17025–17030 (2002).

    Article  CAS  Google Scholar 

  13. Ponjavic, J. et al. Genome Biol. 7, R78 (2006).

    Article  Google Scholar 

  14. Wobbe, C.R. & Struhl, K . Mol. Cell. Biol. 10, 3859–3867 (1990).

    Article  CAS  Google Scholar 

  15. Leach, K.M. et al. Nucleic Acids Res. 31, 1292–1301 (2003).

    Article  CAS  Google Scholar 

  16. Cooper, S.J., Trinklein, N.D., Anton, E.D., Nguyen, L. & Myers, R.M . Genome Res. 16, 1–10 (2006).

    Article  CAS  Google Scholar 

Download references

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.

Author information

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
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Choli Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oren Litvin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. David L Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Dana Pe'er
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jay Shendure
    View author publications

    You can also search for this author in PubMed Google Scholar

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 to Rupali P Patwardhan or Jay Shendure.

Supplementary information

Supplementary Text and Figures

Supplementary Figs. 1–8, Supplementary Tables 1–4, Supplementary Note and Supplementary Methods (PDF 894 kb)

Supplementary Data

Promoter Mutagenesis Data (ZIP 515 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.1589

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing