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

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

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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

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)

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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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