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Fluorogenic DNA sequencing in PDMS microreactors

Nature Methods volume 8, pages 575580 (2011) | Download Citation

Abstract

We developed a multiplex sequencing-by-synthesis method combining terminal phosphate–labeled fluorogenic nucleotides (TPLFNs) and resealable polydimethylsiloxane (PDMS) microreactors. In the presence of phosphatase, primer extension by DNA polymerase using nonfluorescent TPLFNs generates fluorophores, which are confined in the microreactors and detected. We immobilized primed DNA templates in the microreactors, then sequentially introduced one of the four identically labeled TPLFNs, sealed the microreactors and recorded a fluorescence image after template-directed primer extension. With cycle times of <10 min, we demonstrate 30 base reads with 99% raw accuracy. Our 'fluorogenic pyrosequencing' offers benefits of pyrosequencing, such as rapid turnaround, one-color detection and generation of native DNA, along with high detection sensitivity and simplicity of parallelization because simultaneous real-time monitoring of all microreactors is not required.

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Acknowledgements

This work was supported by US National Institutes of Health National Human Genome Research Institute (HG005097-01) to X.S.X. and US National Institutes of Health National Human Genome Research Institute Recovery Act Grand Opportunities grant (1RC2HG005613-01) to X.S.X. W.J.G. is a Damon Runyon Fellow supported by the Damon Runyon Cancer Research Foundation (DRG-2000 09). We acknowledge J. Foley, S. Song, L. Song, G. Holtom and K. Fiala for valuable discussions and technical assistance. This work was performed in part at the Center for Nanoscale Systems, which is a part of Harvard University and a member of the National Nanotechnology Infrastructure Network, which is supported by the National Science Foundation award ECS-0335765.

Author information

Author notes

    • Peter A Sims
    •  & William J Greenleaf

    These authors contributed equally to this work.

Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA.

    • Peter A Sims
    • , William J Greenleaf
    • , Haifeng Duan
    •  & X Sunney Xie

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Contributions

P.A.S., W.J.G. and X.S.X. conceived the fluorogenic pyrosequencing concept. P.A.S. and W.J.G. constructed the sequencing apparatus. H.D. synthesized the TPLFNs. P.A.S. collected and analyzed the data. W.J.G. carried out the microfabrication. P.A.S., W.J.G., H.D. and X.S.X. wrote the manuscript.

Competing interests

Harvard University has filed patent applications based on this work.

Corresponding author

Correspondence to X Sunney Xie.

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    Supplementary Text and Figures

    Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Notes 1–3

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DOI

https://doi.org/10.1038/nmeth.1629

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