Publication Date:
2008-11-22
Description:
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.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Eid, John -- Fehr, Adrian -- Gray, Jeremy -- Luong, Khai -- Lyle, John -- Otto, Geoff -- Peluso, Paul -- Rank, David -- Baybayan, Primo -- Bettman, Brad -- Bibillo, Arkadiusz -- Bjornson, Keith -- Chaudhuri, Bidhan -- Christians, Frederick -- Cicero, Ronald -- Clark, Sonya -- Dalal, Ravindra -- Dewinter, Alex -- Dixon, John -- Foquet, Mathieu -- Gaertner, Alfred -- Hardenbol, Paul -- Heiner, Cheryl -- Hester, Kevin -- Holden, David -- Kearns, Gregory -- Kong, Xiangxu -- Kuse, Ronald -- Lacroix, Yves -- Lin, Steven -- Lundquist, Paul -- Ma, Congcong -- Marks, Patrick -- Maxham, Mark -- Murphy, Devon -- Park, Insil -- Pham, Thang -- Phillips, Michael -- Roy, Joy -- Sebra, Robert -- Shen, Gene -- Sorenson, Jon -- Tomaney, Austin -- Travers, Kevin -- Trulson, Mark -- Vieceli, John -- Wegener, Jeffrey -- Wu, Dawn -- Yang, Alicia -- Zaccarin, Denis -- Zhao, Peter -- Zhong, Frank -- Korlach, Jonas -- Turner, Stephen -- R01HG003710/HG/NHGRI NIH HHS/ -- New York, N.Y. -- Science. 2009 Jan 2;323(5910):133-8. doi: 10.1126/science.1162986. Epub 2008 Nov 20.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Pacific Biosciences, 1505 Adams Drive, Menlo Park, CA 94025, USA.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/19023044" target="_blank"〉PubMed〈/a〉
Keywords:
Base Sequence
;
Consensus Sequence
;
DNA/biosynthesis
;
DNA, Circular/chemistry
;
DNA, Single-Stranded/chemistry
;
DNA-Directed DNA Polymerase/*metabolism
;
Deoxyribonucleotides/metabolism
;
Enzymes, Immobilized
;
Fluorescent Dyes
;
Kinetics
;
Nanostructures
;
Sequence Analysis, DNA/*methods
;
Spectrometry, Fluorescence
Print ISSN:
0036-8075
Electronic ISSN:
1095-9203
Topics:
Biology
,
Chemistry and Pharmacology
,
Computer Science
,
Medicine
,
Natural Sciences in General
,
Physics
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