Real-Time DNA Sequencing from Single Polymerase Molecules

被引：2534

作者：

Eid, John ^{[1
]}

Fehr, Adrian ^{[1
]}

Gray, Jeremy ^{[1
]}

Luong, Khai ^{[1
]}

Lyle, John ^{[1
]}

Otto, Geoff ^{[1
]}

Peluso, Paul ^{[1
]}

Rank, David ^{[1
]}

Baybayan, Primo ^{[1
]}

Bettman, Brad ^{[1
]}

Bibillo, Arkadiusz ^{[1
]}

Bjornson, Keith ^{[1
]}

Chaudhuri, Bidhan ^{[1
]}

Christians, Frederick ^{[1
]}

Cicero, Ronald ^{[1
]}

Clark, Sonya ^{[1
]}

Dalal, Ravindra ^{[1
]}

deWinter, Alex ^{[1
]}

Dixon, John ^{[1
]}

Foquet, Mathieu ^{[1
]}

Gaertner, Alfred ^{[1
]}

Hardenbol, Paul ^{[1
]}

Heiner, Cheryl ^{[1
]}

Hester, Kevin ^{[1
]}

Holden, David ^{[1
]}

Kearns, Gregory ^{[1
]}

Kong, Xiangxu ^{[1
]}

Kuse, Ronald ^{[1
]}

Lacroix, Yves ^{[1
]}

Lin, Steven ^{[1
]}

Lundquist, Paul ^{[1
]}

Ma, Congcong ^{[1
]}

Marks, Patrick ^{[1
]}

Maxham, Mark ^{[1
]}

Murphy, Devon ^{[1
]}

Park, Insil ^{[1
]}

Pham, Thang ^{[1
]}

Phillips, Michael ^{[1
]}

Roy, Joy ^{[1
]}

Sebra, Robert ^{[1
]}

Shen, Gene ^{[1
]}

Sorenson, Jon ^{[1
]}

Tomaney, Austin ^{[1
]}

Travers, Kevin ^{[1
]}

Trulson, Mark ^{[1
]}

Vieceli, John ^{[1
]}

Wegener, Jeffrey ^{[1
]}

Wu, Dawn ^{[1
]}

Yang, Alicia ^{[1
]}

Zaccarin, Denis ^{[1
]}

机构：

[1] Pacific Biosci, Menlo Pk, CA 94025 USA

来源：

SCIENCE | 2009年 / 323卷 / 5910期

关键词：

PHOSPHATE-LABELED NUCLEOTIDES; HUMAN GENOME; MODE; FLUORESCENCE; SPECTROSCOPY; RESOLUTION; ALGORITHM; SUBSTRATE;

D O I：

10.1126/science.1162986

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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.

引用

页码：133 / 138

页数：6

共 40 条

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