September 21, 2019  |  

Real-time DNA sequencing from single polymerase molecules.

Authors: Eid, John and Fehr, Adrian and Gray, Jeremy and Luong, Khai and Lyle, John and Otto, Geoff and Peluso, Paul and Rank, David and Baybayan, Primo and Bettman, Brad and Bibillo, Arkadiusz and Bjornson, Keith and Chaudhuri, Bidhan and Christians, Frederick and Cicero, Ronald and Clark, Sonya and Dalal, Ravindra and Dewinter, Alex and Dixon, John and Foquet, Mathieu and Gaertner, Alfred and Hardenbol, Paul and Heiner, Cheryl and Hester, Kevin and Holden, David and Kearns, Gregory and Kong, Xiangxu and Kuse, Ronald and Lacroix, Yves and Lin, Steven and Lundquist, Paul and Ma, Congcong and Marks, Patrick and Maxham, Mark and Murphy, Devon and Park, Insil and Pham, Thang and Phillips, Michael and Roy, Joy and Sebra, Robert and Shen, Gene and Sorenson, Jon and Tomaney, Austin and Travers, Kevin and Trulson, Mark and Vieceli, John and Wegener, Jeffrey and Wu, Dawn and Yang, Alicia and Zaccarin, Denis and Zhao, Peter and Zhong, Frank and Korlach, Jonas and Turner, Stephen

We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

Journal: Science
DOI: 10.1126/science.1162986
Year: 2009

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