Real-Time DNA Sequencing from Single Polymerase Molecules Export

@article{citeulike:3623940, abstract = {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. 10.1126/science.1162986}, author = {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}, citeulike-article-id = {3623940}, citeulike-linkout-0 = {http://dx.doi.org/10.1126/science.1162986}, citeulike-linkout-1 = {http://www.sciencemag.org/content/323/5910/133.abstract}, citeulike-linkout-2 = {http://www.sciencemag.org/content/323/5910/133.full.pdf}, citeulike-linkout-3 = {http://www.sciencemag.org/cgi/content/abstract/1162986//}, citeulike-linkout-4 = {http://view.ncbi.nlm.nih.gov/pubmed/19023044}, citeulike-linkout-5 = {http://www.hubmed.org/display.cgi?uids=19023044}, day = {2}, doi = {10.1126/science.1162986}, issn = {1095-9203}, journal = {Science}, keywords = {clip2, dna-polymerase, experiment, fluorescent, single\_molecule, visualization}, month = {January}, number = {5910}, pages = {133--138}, posted-at = {2009-01-20 06:21:51}, priority = {2}, title = {Real-Time DNA Sequencing from Single Polymerase Molecules}, url = {http://dx.doi.org/10.1126/science.1162986}, volume = {323}, year = {2009} }

TY - JOUR ID - citeulike:3623940 L3 - citeulike-article-id:3623940 N2 - 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. 10.1126/science.1162986 IS - 5910 JF - Science SN - 1095-9203 EP - 138 TI - Real-Time DNA Sequencing from Single Polymerase Molecules VL - 323 SP - 133 KW - clip2 KW - dna-polymerase KW - experiment KW - fluorescent KW - single_molecule KW - visualization AU - Eid, John AU - Fehr, Adrian AU - Gray, Jeremy AU - Luong, Khai AU - Lyle, John AU - Otto, Geoff AU - Peluso, Paul AU - Rank, David AU - Baybayan, Primo AU - Bettman, Brad AU - Bibillo, Arkadiusz AU - Bjornson, Keith AU - Chaudhuri, Bidhan AU - Christians, Frederick AU - Cicero, Ronald AU - Clark, Sonya AU - Dalal, Ravindra AU - deWinter, Alex AU - Dixon, John AU - Foquet, Mathieu AU - Gaertner, Alfred AU - Hardenbol, Paul AU - Heiner, Cheryl AU - Hester, Kevin AU - Holden, David AU - Kearns, Gregory AU - Kong, Xiangxu AU - Kuse, Ronald AU - Lacroix, Yves AU - Lin, Steven AU - Lundquist, Paul AU - Ma, Congcong AU - Marks, Patrick AU - Maxham, Mark AU - Murphy, Devon AU - Park, Insil AU - Pham, Thang AU - Phillips, Michael AU - Roy, Joy AU - Sebra, Robert AU - Shen, Gene AU - Sorenson, Jon AU - Tomaney, Austin AU - Travers, Kevin AU - Trulson, Mark AU - Vieceli, John AU - Wegener, Jeffrey AU - Wu, Dawn AU - Yang, Alicia AU - Zaccarin, Denis AU - Zhao, Peter AU - Zhong, Frank AU - Korlach, Jonas AU - Turner, Stephen PY - 2009/01/02/ UR - http://dx.doi.org/10.1126/science.1162986 ER -