Real-Time DNA Sequencing from Single Polymerase Molecules

作者：John S. Eid, Adrian Fehr, Jeremy Gray, Khai Luong, John M. Lyle, Geoff A. Otto, Paul S. Peluso, David R. Rank, Primo A. Baybayan, Brad Bettman, Arkadiusz Bibiłło, Keith P. Bjornson, Bidhan Chaudhuri, Fred C. Christians, Ronald L. Cicero, Sonya Clark, Ravindra V. Dalal, Alex D. deWinter, John W. Dixon, Mathieu Foquet, Alfred L. Gaertner, Paul Hardenbol, Cheryl R. Heiner, Kevin Hester, David W. Holden, Gregory L. Kearns, Xiangxu Kong, Ronald Kuse, Yves Lacroix, Steven Lin, Paul M. Lundquist, Congcong Ma, Patrick J. Marks, Mark Maxham, Devon L. Murphy, Insil Park, Thang Pham, Michael Phillips, Joy Kumar Roy, Robert Sebra, Gene Shen, Jon M. Sorenson, Austin B. Tomaney, Kevin J. Travers, Mark O. Trulson, John Vieceli, Jeffrey Wegener, Dawn Wu, Alicia Yang, Denis Zaccarin, Peter Y. Zhao, Frank W. Zhong, Jonas Korlach, Stephen Turner · 发表于：Science · 年份：2008 · DOI：10.1126/science.1162986 · 被引用次数：3886 · 研究领域：Advanced biosensing and bioanalysis techniques、Genomics and Phylogenetic Studies、Biosensors and Analytical Detection

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.