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Contributed by Nicholas J. Turro, April 28, 2008 (sent for review March 21, 2008)
(ddCT P-N 3 -Bodipy-FL-510, ddUT P-N 3 -R6G, ddAT P-N 3 ROX, and ddGTP-N3-Cy5) (Fig. 2) are incorporated accurately
in a base-specific manner in a polymerase reaction, single-base
extension reactions with four different self-priming DNA templates whose next complementary base was either A, C, G, or T
Guo et al.
were carried out in solution. After the reaction, the four different
primer extension products were analyzed by MALDI-TOF MS
as shown in Fig. 4. Single clear mass peaks at 9,180, 8,915, 9,317,
and 9,082 (m/z) corresponding to each primer extension product
with no leftover starting materials were produced by using
ddNTP-N3-fluorophores (Fig. 4 A, C, E, and G). Brief incubation of the DNA extension products in an aqueous TCEP
solution led to the cleavage of the linker tethering the fluorophore to the dideoxynucleotide. Fig. 4 B, D, F, and H shows the
cleavage results for the DNA products extended with ddNTPN3-fluorophores. The mass peaks at 9,180, 8,915, 9,317, and
9,082 (m/z) have completely disappeared, whereas single peaks
corresponding to the cleavage products appear at 8,417, 8,394,
8,433, and 8,395 (m/z), respectively. These results demonstrate
that cleavable fluorescent ddNTPs are successfully synthesized
and efficiently terminated the DNA synthesis in a polymerase
reaction and that the fluorophores are quantitatively cleaved by
TCEP. Thus, these ddNTP analogues meet the key requirements
necessary for performing the hybrid SBS in combination with the
NRTs.
Four-Color DNA Sequencing on a Chip by Using Cleavable Fluorescent
Dideoxynucleotides and 3-O-Modified NRTs by the Hybrid SBS Approach. In our four-color hybrid SBS approach, the identity of the
incorporated nucleotide is determined by the unique fluorescence emission from the four fluorescent dideoxynucleotides,
whereas the role of the 3-O-modified NRTs is to further extend
the DNA strand. Therefore, the ratio of the ddNTP-N3fluorophores and 3-O-N3-dNTPs during the polymerase reaction determines how much of the ddNTP-N3-fluorophores incorporate and, thus, the corresponding fluorescence emission
strength. With a finite amount of immobilized DNA template on
a solid surface, initially the majority of the priming strands
should be extended with 3-O-N3-dNTPs, whereas a relatively
smaller amount should be extended with ddNTP-N3-fluoroPNAS July 8, 2008 vol. 105 no. 27 9147
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Fig. 2. Structures of the cleavable fluorescent ddNTPs: ddCTP-N3-Bodipy-FL510 (abs (max) 502 nm; em (max) 510 nm), ddUTP-N3-R6G (abs (max) 525 nm;
em (max) 550 nm), ddATP-N3-ROX (abs (max) 585 nm; em (max) 602 nm), and
ddGTP-N3-Cy5 (abs (max) 649 nm; em (max) 670 nm).
Fig. 4. A polymerase reaction scheme (Top) to yield DNA extension products by incorporating each of the four ddNTP-N3-fluorophores and the subsequent
cleavage reaction to remove the fluorophores from the DNA extension products. MALDI-TOF MS spectra (Bottom) showing efficient base-specific incorporation
of the ddNTP-N3-fluorophores and the subsequent cleavage of the fluorophores from the DNA extension products: (A) Primer extended with ddATP-N3-ROX (1)
(peak at 9,180 m/z), (B) its cleavage product 2 (8,417 m/z); (C) primer extended with ddCTP-N3-Bodipy-FL-510 (3) (peak at 8,915 m/z), (D) its cleavage product 4
(8,394 m/z); (E) primer extended with ddGTP-N3-Cy5 (5) (peak at 9,317 m/z), (F) its cleavage product 6 (8,433 m/z); (G) primer extended with ddUTP-N3-R6G (7)
(peak at 9,082 m/z), and (H) its cleavage product 8 (8,395 m/z).
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Fig. 5. Four-color DNA sequencing by the hybrid SBS. (A) A hybrid SBS scheme for four-color sequencing on a chip by using four 3-O-N3-dNTPs and four
ddNTP-N3-fluorophores. (B) The four-color fluorescence images for each step of the SBS: (1) incorporation of 3-O-N3-dCTP and ddCTP-N3-Bodipy-FL-510; (2)
cleavage of N3-Bodipy-FL-510 and 3-CH2N3 group; (3) incorporation of 3-O-N3-dATP and ddATP-N3-Rox; (4) cleavage of N3-ROX and 3-CH2N3 group; (5)
incorporation of 3-O-N3-dTTP and ddUTP-N3-R6G; (6) cleavage of N3-R6G and 3-CH2N3 group; (7) incorporation of 3-O-N3-dGTP and ddGTP-N3-Cy5; (8) cleavage
of N3-Cy5 and 3-CH2N3 group; images 9 63 are similarly produced. (C) A plot (four-color sequencing data) of raw fluorescence emission intensity obtained by
using 3-O-N3-dNTPs and ddNTP-N3-fluorophores. The small groups of peaks between the identified bases are fluorescent background from the DNA chip.
1. Lander ES, et al. (2001) Initial sequencing and analysis of the human genome. Nature
409:860 921.
2. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating
inhibitors. Proc Natl Acad Sci USA 74:54635467.
3. Smith LM, et al. (1986) Fluorescence detection in automated DNA sequence analysis.
Nature 321:674 679.
4. Prober JM, et al. (1987) A system for rapid DNA sequencing with fluorescent chainterminating dideoxynucleotides. Science 238:336 341.
5. Ju J, Ruan C, Fuller CW, Glazer AN, Mathies RA (1995) Fluorescence energy transfer dye-labeled
primers for DNA sequencing and analysis. Proc Natl Acad Sci USA 92:43474351.
6. Blazej RG, Kumaresan P, Mathies RA (2006) Microfabricated bioprocessor for integrated nanoliter-scale Sanger DNA sequencing. Proc Natl Acad Sci USA 103:7240 7245.
7. Drmanac S, et al. (1998) Accurate sequencing by hybridization for DNA diagnostics and
individual genomics. Nat Biotechnol 16:54 58.
8. Fu DJ, et al. (1998) Sequencing exons 5 to 8 of the p53 gene by MALDI-TOF mass
spectrometry. Nat Biotechnol 16:381384.
9. Edwards JR, Itagaki Y, Ju J (2001) DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry. Nucleic Acids Res 29:E104.
10. Kasianowicz JJ, Brandin E, Branton D, Deamer DW (1996) Characterization of individual polynucleotide molecules using a membrane channel. Proc Natl Acad Sci USA
93:13770 13773.
11. Shendure J, et al. (2005) Accurate multiplex polony sequencing of an evolved bacterial
genome. Science 309:1728 1732.
12. Ronaghi M, Uhlen M, Nyren P (1998) A sequencing method based on real-time
pyrophosphate. Science 281:363365.
13. Harris TD, et al. (2008) Single-molecule DNA sequencing of a viral genome. Science
320:106 109.
14. Greenleaf WJ, Block SM (2006) Single-molecule, motion-based DNA sequencing using
RNA polymerase. Science 313:801.
15. Mitra RD, Shendure J, Olejnik J, Olejnik EK, Church GM (2003) Fluorescent in situ
sequencing on polymerase colonies. Anal Biochem 320:55 65.
16. Ju J, Li Z, Edwards J, Itagaki Y (2003) Massive parallel method for decoding DNA and
RNA. US Patent 6,664,079.
17. Li Z, et al. (2003) A photocleavable fluorescent nucleotide for DNA sequencing and
analysis. Proc Natl Acad Sci USA 100:414 419.
18. Ruparel H, et al. (2005) Design and synthesis of a 3-O-allyl photocleavable fluorescent
nucleotide as a reversible terminator for DNA sequencing by synthesis. Proc Natl Acad
Sci USA 102:59325937.
9150 www.pnas.orgcgidoi10.1073pnas.0804023105
19. Seo TS, et al. (2005) Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. Proc Natl Acad Sci USA 102:5926 5931.
20. Ju J, et al. (2006) Four-color DNA sequencing by synthesis using cleavable fluorescent
nucleotide reversible terminators. Proc Natl Acad Sci USA 103:1963519640.
21. Mikkelsen TS, et al. (2007) Genome-wide maps of chromatin state in pluripotent and
lineage-committed cells. Nature 448:553560.
22. Johnson DS, Mortazavi A, Myers RM, Wold B (2007) Genome-wide mapping of in vivo
protein-DNA interactions. Science 316:14971502.
23. Barski A, et al. (2007) High-resolution profiling of histone methylations in the human
genome. Cell 129:823 837.
24. Wu J, et al. (2007) 3-O-modified nucleotides as reversible terminators for pyrosequencing. Proc Natl Acad Sci USA 104:1646216467.
25. Zavgorodny S, et al. (1991) 1-Alkylthioalkylation of nucleoside hydroxyl functions and
its synthetic applications: A new versatile method in nucleoside chemistry. Tetrahedron Lett 32:75937596.
26. Zavgorodny S, Pechenov AE, Shvets VI, Miroshnikov AI (2000) S,X-acetals in nucleoside
chemistry. III. Synthesis of 2-and 3-O-azidomethyl derivatives of ribonucleosides.
Nucleosides, Nucleotides Nucleic Acids 19:19771991.
27. Barnes C, Balasubramanian S, Liu X, Swerdlow H, Milton J (2006) Labelled nucleotides.
US Patent 7,057,026.
28. Saxon E, Bertozzi CR (2000) Cell surface engineering by a modified Staudinger reaction.
Science 287:20072010.
29. Milton J, Ruediger S, Liu X (2006) Nucleosides/nucleotides conjugated to labels via
cleavable linkages and their use in nucleic acid sequencing. US Patent Appl
US20060160081A1.
30. Rosenblum BB, et al. (1997) New dye-labeled terminators for improved DNA sequencing patterns. Nucleic Acids Res 25:4500 4504.
31. Wu W, et al. (2007) Termination of DNA synthesis by N6-alkylated, not 3-Oalkylated, photocleavable 2-deoxyadenosine triphosphates. Nucleic Acids Res
35:6339 6349.
32. Velculescu VE, Zhang L, Vogelstein B, Kinzler KW (1995) Serial analysis of gene
expression. Science 270:484 487.
33. Kim JB, et al. (2007) Polony multiplex analysis of gene expression (PMAGE) in mouse
hypertrophic cardiomyopathy. Science 316:14811484.
34. Margulies M, et al. (2005) Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376 380.
Guo et al.