Abstract
We present dial-out PCR, a highly parallel method for retrieving accurate DNA molecules for gene synthesis. A complex library of DNA molecules is modified with unique flanking tags before massively parallel sequencing. Tag-directed primers then enable the retrieval of molecules with desired sequences by PCR. Dial-out PCR enables multiplex in vitro clone screening and is a compelling alternative to in vivo cloning and Sanger sequencing for accurate gene synthesis.
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Acknowledgements
We thank J.B. Hiatt, J.O. Kitzman, R.P. Patwardhan, J. Cooper, L. Pennacchio, E. Rubin and S. Deutsch for helpful discussions. We also thank Y. Zhang (Albert Einstein College of Medicine) for providing the SLiCE strain and R. Qiu for preparing the SLiCE extract. J.J.S. was funded by a Helen Hay Whitney Foundation postdoctoral fellowship. Our work was supported in part by a grant from the US National Institutes of Health–National Cancer Institute (1R21CA160080 to J.S.).
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J.J.S. and J.S. designed the research, J.J.S. performed the research, C.L. sequenced the libraries, and J.J.S. and J.S. analyzed the data and wrote the paper.
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J.J.S. and J.S. are authors of a patent application for the method described in this paper (US Provisional Application number 61/478,016).
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Supplementary Figures 1–5, Supplementary Tables 1–5 and Supplementary Notes 1–3 (PDF 2226 kb)
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Schwartz, J., Lee, C. & Shendure, J. Accurate gene synthesis with tag-directed retrieval of sequence-verified DNA molecules. Nat Methods 9, 913–915 (2012). https://doi.org/10.1038/nmeth.2137
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DOI: https://doi.org/10.1038/nmeth.2137
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