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Fluorogenic DNA sequencing in PDMS microreactors

Nature Methods volume 8, pages 575–580 (2011)Cite this article

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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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Figure 1: Fluorogenic pyrosequencing chemistry and workflow.
Figure 2: Schematic of the fluorogenic pyrosequencing system.
Figure 3: Fluorogenic pyrosequencing images and homopolymer analysis.
Figure 4: Sequencing traces of quasi-random sequences and analysis of global errors.

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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.

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  1. Peter A Sims and William J Greenleaf: These authors contributed equally to this work.

Authors and 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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  1. Peter A Sims
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  2. William J Greenleaf
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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.

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Correspondence to X Sunney Xie.

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Harvard University has filed patent applications based on this work.

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Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Notes 1–3 (PDF 534 kb)

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Sims, P., Greenleaf, W., Duan, H. et al. Fluorogenic DNA sequencing in PDMS microreactors. Nat Methods 8, 575–580 (2011). https://doi.org/10.1038/nmeth.1629

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