Real-Time DNA Sequencing from Single Polymerase Molecules
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.
Get full access to this article
View all available purchase options and get full access to this article.
Supplementary Material
References and Notes
1
F. Sanger, S. Nicklen, A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A.74, 5463 (1977).
2
L. Blancoet al., J. Biol. Chem.264, 8935 (1989).
3
A. Kornberg, T. A. Baker, DNA Replication (Freeman, New York, ed. 2, 1992).
4
S. Tabor, H. E. Huber, C. C. Richardson, J. Biol. Chem.262, 16212 (1987).
5
C. Feschotte, E. J. Pritham, Annu. Rev. Genet.41, 331 (2007).
6
E. Tuzunet al., Nat. Genet.37, 727 (2005).
7
S. Balasubramanian, D. R. Bentley, Patent WO 01/057248 (2001).
8
I. Braslavsky, B. Hebert, E. Kartalov, S. R. Quake, Proc. Natl. Acad. Sci. U.S.A.100, 3960 (2003).
9
M. Ronaghi, M. Uhlen, P. Nyren, Science281, 363 (1998).
10
J. Shendureet al., Science309, 1728 (2005).
11
D. R. Bentley, Curr. Opin. Genet. Dev.16, 545 (2006).
12
M. L. Metzker, Genome Res.15, 1767 (2005).
13
J. B. Fanet al., Methods Enzymol.410, 57 (2006).
14
T. D. Harriset al., Science320, 106 (2008).
15
M. Margulieset al., Nature437, 376 (2005).
16
A. Valouevet al., Genome Res.18, 1051 (2008).
17
E. Y. Chan, U.S. Patent 6,210,896 (2001).
18
S. L. Cockroft, J. Chu, M. Amorin, M. R. Ghadiri, J. Am. Chem. Soc.130, 818 (2008).
19
W. J. Greenleaf, S. M. Block, Science313, 801 (2006).
20
M. J. Leveneet al., Science299, 682 (2003).
21
B. A. Mulderet al., Nucleic Acids Res.33, 4865 (2005).
22
B. Reynolds, R. Miller, J. G. Williams, J. P. Anderson, Nucleosides Nucleotides Nucleic Acids27, 18 (2008).
23
M. Foquetet al., J. Appl. Phys.103, 034301 (2008).
24
M. J. Lang, P. M. Fordyce, A. M. Engh, K. C. Neuman, S. M. Block, Nat. Methods1, 133 (2004).
25
A. M. Lieto, R. C. Cush, N. L. Thompson, Biophys. J.85, 3294 (2003).
26
See supporting material on Science Online.
27
J. Juet al., Proc. Natl. Acad. Sci. U.S.A.103, 19635 (2006).
28
R. D. Mitra, J. Shendure, J. Olejnik, O. Edyta Krzymanska, G. M. Church, Anal. Biochem.320, 55 (2003).
29
C. C. Kao, T. Widlanski, W. Vassiliou, J. Epp, U.S. Patent 6,399,335 (2002).
30
S. Kumaret al., Nucleosides Nucleotides Nucleic Acids24, 401 (2005).
31
A. Soodet al., J. Am. Chem. Soc.127, 2394 (2005).
32
J. Korlachet al., Nucleosides Nucleotides Nucleic Acids27, 1072 (2008).
33
F. B. Deanet al., Proc. Natl. Acad. Sci. U.S.A.99, 5261 (2002).
34
J. Korlachet al., Proc. Natl. Acad. Sci. U.S.A.105, 1176 (2008).
35
P. M. Lundquistet al., Opt. Lett.33, 1026 (2008).
36
C. Castroet al., Proc. Natl. Acad. Sci. U.S.A.104, 4267 (2007).
37
K. Horne, Publ. Astron. Soc. Pac.98, 609 (1986).
38
O. Gotoh, J. Mol. Biol.162, 705 (1982).
39
D. Gusfield, Algorithms on Strings, Trees, and Sequences: Computer Science and Computational Biology (Cambridge Univ. Press, Cambridge, 1997).
40
J. T. Bosierset al., Proc. SPIE6996, 69960Z (2008).
41
A. J. P. Theuwissen, Solid State Electron.52, 1401 (2008).
42
A. J. Bermanet al., EMBO J.26, 3494 (2007).
43
We thank the entire staff at Pacific Biosciences, and J. Puglisi, M. Hunkapiller, R. Kornberg, K. Johnson, D. Haussler, W. Webb, and H. Craighead for many helpful discussions. Supported by National Human Genome Research Institute grant R01HG003710.
(0)eLetters
eLetters is a forum for ongoing peer review. eLetters are not edited, proofread, or indexed, but they are screened. eLetters should provide substantive and scholarly commentary on the article. Embedded figures cannot be submitted, and we discourage the use of figures within eLetters in general. If a figure is essential, please include a link to the figure within the text of the eLetter. Please read our Terms of Service before submitting an eLetter.
Log In to Submit a ResponseNo eLetters have been published for this article yet.
Information & Authors
Information
Published In
Science
Volume 323 | Issue 5910
2 January 2009
2 January 2009
Copyright
American Association for the Advancement of Science.
Article versions
You are viewing the most recent version of this article.
Submission history
Received: 9 July 2008
Accepted: 20 October 2008
Published in print: 2 January 2009
Notes
Supporting Online Material
www.sciencemag.org/cgi/content/full/1162986/DC1
Materials and Methods
Figs. S1 to S8
Tables S1 to S3
Movie S1
References
Authors
Metrics & Citations
Metrics
Article Usage
Altmetrics
Citations
Cite as
- John Eid et al.
Export citation
Select the format you want to export the citation of this publication.
Cited by
View Options
Check Access
Log in to view the full text
AAAS login provides access to Science for AAAS Members, and access to other journals in the Science family to users who have purchased individual subscriptions.
- Become a AAAS Member
- Activate your AAAS ID
- Purchase Access to Other Journals in the Science Family
- Account Help
Log in via OpenAthens.
Log in via Shibboleth.
More options
Register for free to read this article
As a service to the community, this article is available for free. Login or register for free to read this article.
Buy a single issue of Science for just $15 USD.
An approach to increase genome sequencing coverage
As stressful genes are common in genomes of various creatures, numerous genes cannot be amplified, cloned or expressed. Some modified bases can be subjected to breakage and damage in laboratory manipulations. Many gaps remain undetermined after whole genome sequencing. A method involving gentle cell lysis with ethanol, DNA denaturation in over 50% formamide or 2-pyrrolidone (1) at about 50 to 60˚C, and subsequent DNA synthesis with a viral DNA polymerase might produce intact DNA or PCR templates for next generation and third generation sequencing. This procedure could increase genome sequencing coverage, as it circumvents DNA breakage caused by high temperature and chemical reagents. Some viral DNA polymerases are capable of recognizing various DNA modifications as viruses are prone to mutations in the hosts.
Yan Huang, 2 Xiaoyi Hu, 3 Zhumei He, 1 Qiuyun Liu 1*
1. State Key Laboratory of Biocontrol, Key Laboratory for Improved Variety Reproduction of Aquatic Economic Animals of Guangdong Province, Biotechnology Research Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
2. Academy building, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong.
3. Department of Chemical and Biomolecular Engineering, New York University Tandon School of Engineering, 6 Metro Tech Center, Brooklyn, NY 11201, USA.
*Corresponding author. Email: [email protected] (Q. Liu)
References
1.R. Chakrabarti, C. E. Schutt. Nucleic Acids Res 29, 2377 (2001).
ACKNOWLEDGMENT
Manuscript editing by Ms. Yan Shi is appreciated.