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`APPLIEDBIOLOGICAL
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`Four-color DNA sequencing with 3ⴕ-O-modified
`nucleotide reversible terminators and chemically
`cleavable fluorescent dideoxynucleotides
`
`Jia Guo*†‡, Ning Xu*, Zengmin Li*†, Shenglong Zhang*†‡, Jian Wu*†‡, Dae Hyun Kim*, Mong Sano Marma*†,
`Qinglin Meng*†‡, Huanyan Cao*, Xiaoxu Li*†, Shundi Shi*, Lin Yu*, Sergey Kalachikov*, James J. Russo*,
`Nicholas J. Turro†‡§, and Jingyue Ju*†§
`
`*Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Departments of †Chemical Engineering and
`‡Chemistry, Columbia University, New York, NY 10027
`
`Contributed by Nicholas J. Turro, April 28, 2008 (sent for review March 21, 2008)
`
`DNA sequencing by synthesis (SBS) on a solid surface during
`polymerase reaction can decipher many sequences in parallel. We
`report here a DNA sequencing method that is a hybrid between the
`Sanger dideoxynucleotide terminating reaction and SBS. In this
`approach, four nucleotides, modified as reversible terminators by
`capping the 3ⴕ-OH with a small reversible moiety so that they are
`still recognized by DNA polymerase as substrates, are combined
`with four cleavable fluorescent dideoxynucleotides to perform
`SBS. The ratio of the two sets of nucleotides is adjusted as the
`extension cycles proceed. Sequences are determined by the unique
`fluorescence emission of each fluorophore on the DNA products
`terminated by ddNTPs. On removing the 3ⴕ-OH capping group from
`the DNA products generated by incorporating the 3ⴕ-O-modified
`dNTPs and the fluorophore from the DNA products terminated
`with the ddNTPs, the polymerase reaction reinitiates to continue
`the sequence determination. By using an azidomethyl group as a
`chemically reversible capping moiety in the 3ⴕ-O-modified dNTPs,
`and an azido-based cleavable linker to attach the fluorophores to
`the ddNTPs, we synthesized four 3ⴕ-O-azidomethyl-dNTPs and four
`ddNTP-azidolinker-fluorophores for the hybrid SBS. After se-
`quence determination by fluorescence imaging, the 3ⴕ-O-azidom-
`ethyl group and the fluorophore attached to the DNA extension
`product via the azidolinker are efficiently removed by using Tris(2-
`carboxyethyl)phosphine in aqueous solution that is compatible
`with DNA. Various DNA templates, including those with homopoly-
`mer regions, were accurately sequenced with a read length of >30
`bases by using this hybrid SBS method on a chip and a four-color
`fluorescence scanner.
`
`sequencing by synthesis 兩 DNA chip
`
`The completion of the Human Genome Project (1) was a
`
`monumental achievement in biological science. The engine
`behind this project was the Sanger sequencing method (2), which
`is still the gold standard in genome research. The prolonged
`success of the Sanger sequencing method is because of its
`efficiency and fidelity in producing dideoxy-terminated DNA
`products that can be separated electrophoretically and detected
`by fluorescence (3–5). However, a challenge in the use of
`electrophoresis for DNA separation is the difficulty in achieving
`high throughput and the complexity involved in the automation,
`although some level of increased parallelization may be achieved
`by using miniaturization (6).
`To overcome the limitations of the Sanger sequencing tech-
`nology, a variety of new methods have been investigated. Such
`approaches include sequencing by hybridization (7), mass spec-
`trometry sequencing (8, 9), sequencing by nanopores (10), and
`sequencing by ligation (11). More recently, DNA sequencing by
`synthesis (SBS) approaches such as pyrosequencing (12), se-
`quencing of single DNA molecules (13, 14), and polymerase
`colonies (15) have been widely explored. Previously, we reported
`the development of a general strategy to rationally design
`
`cleavable fluorescent nucleotide reversible terminators (NRTs)
`for four-color DNA sequencing by synthesis (16–20). In this
`approach, four nucleotides (A, C, G and T) are modified as
`NRTs by attaching a cleavable fluorophore to a specific location
`on the base and capping the 3⬘-OH with a small chemically
`reversible moiety so that they are still recognized as substrates
`by DNA polymerase. DNA templates consisting of homopoly-
`mer regions were accurately sequenced by this approach. A
`recently developed SBS system based on a similar design of the
`cleavable fluorescent NRTs has already found wide application
`in genome biology (21–23). In addition, we have used 3⬘-O-
`modified NRTs to solve the homopolymer sequencing problem
`in conventional pyrosequencing (24).
`We report here an alternative sequencing method that is a
`hybrid between the Sanger dideoxy chain-terminating reaction
`and SBS, and discuss the advantages that come with this hybrid
`approach. The fundamental difference between the two methods
`is that the Sanger method produces every possible complemen-
`tary DNA extension fragment for a given DNA template and
`obtains the sequence after the separation and detection of these
`fragments, whereas SBS relies on identification of each base as
`the DNA strand is extended by cleavable fluorescent NRTs that
`temporarily pause the DNA synthesis for sequence determina-
`tion. The limiting factor for increasing throughput in the Sanger
`method is the requirement to use electrophoresis.
`Challenges in using SBS with cleavable fluorescent NRTs
`involve the further improvement of the DNA polymerase that
`efficiently recognizes the modified nucleotides. In addition, the
`first generation of the cleavable fluorescent NRTs synthesize
`the DNA strand with a propargyl amino group modification on
`the base during SBS (20), which might interfere with the activity
`of the polymerase for chain elongation. The advantage of the
`Sanger method is the dideoxy chain fragment-producing reac-
`tion. Once the DNA strand is terminated by incorporation of a
`fluorescent dideoxynucleotide, it is no longer involved in further
`DNA extension reactions. Therefore, the continuous DNA
`polymerase extension reaction occurs with only natural nucle-
`otides at high efficiency leading to a read length of ⬎700 bp. The
`most attractive feature in the SBS approach is the massive
`
`Author contributions: J.G., D.H.K., N.J.T., and J.J. designed research; J.G., N.X., Z.L., S.Z.,
`J.W., D.H.K., Q.M., X.L., S.S., L.Y., and J.J. performed research; Z.L., S.Z., M.S.M., Q.M., H.C.,
`X.L., L.Y., and S.K. contributed new reagents/analytic tools; J.G., N.X., Z.L., S.Z., J.W., D.H.K.,
`Q.M., S.S., J.J.R., and J.J. analyzed data; and J.G., D.H.K., J.J.R., N.J.T., and J.J. wrote the
`paper.
`
`The authors declare no conflict of interest.
`
`Freely available online through the PNAS open access option.
`§To whom correspondence should be addressed. E-mail: njt3@columbia.edu or ju@
`c2b2.columbia.edu.
`
`This article contains supporting information online at www.pnas.org/cgi/content/full/
`0804023105/DCSupplemental.
`
`© 2008 by The National Academy of Sciences of the USA
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804023105
`
`PNAS 兩
`
`July 8, 2008 兩 vol. 105 兩 no. 27 兩 9145–9150
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`Illumina Ex. 1091
`IPR Petition - USP 10,435,742
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`parallel readout capability by using a high-density DNA chip
`without the need to separate the DNA products. We have
`explored the integration of the advantageous features of the two
`methods to develop a hybrid DNA sequencing approach. In this
`method, four nucleotides, modified as reversible terminators by
`capping the 3⬘-OH with a small reversible moiety so that they are
`still recognized as substrates by DNA polymerase, are combined
`with four cleavable fluorescent dideoxynucleotides to perform
`SBS. DNA sequences are determined by the unique fluorescence
`emission of each fluorophore on the DNA products terminated
`by ddNTPs. On removing the 3⬘-OH capping group from the
`DNA products generated by incorporating the 3⬘-O-modified
`dNTPs and the fluorophore from the DNA products termi-
`nated with the ddNTPs, the polymerase reaction reinitiates to
`continue the sequence determination [supporting information
`(SI) Fig. S1].
`By using an azidomethyl group as a chemically reversible
`capping moiety in the 3⬘-O-modified dNTPs, and an azido-based
`cleavable linker to attach the fluorophores to ddNTPs, we
`synthesized four 3⬘-O-azidomethyl-dNTPs (3⬘-O-N3-dNTPs)
`and four ddNTP-azidolinker-f luorophores (ddNTP-N3-
`fluorophores) for the hybrid SBS. The azidomethyl capping
`moiety on the 3⬘-OH group and the cleavable fluorophore on the
`DNA extension products are efficiently removed after fluores-
`cence detection for sequence determination by using a chemical
`method that is compatible with DNA. Various DNA templates,
`including those with homopolymer regions, were accurately
`sequenced with a read length of ⬎30 bases by using this hybrid
`SBS method.
`
`Results and Discussion
`Design and Synthesis of 3ⴕ-O-Modified NRTs and Cleavable Fluorescent
`Dideoxynucleotide Terminators for the Hybrid SBS. A critical re-
`quirement for using SBS methods to unambiguously sequence
`DNA is a suitable chemical moiety to cap the 3⬘-OH of the
`nucleotide such that it temporarily terminates the polymerase
`reaction to allow the identification of the incorporated nucleo-
`tide. A stepwise addition of separate nucleotides with a free
`3⬘-OH group has inherent difficulties in detecting sequences in
`homopolymeric regions (12, 13). Capping the 3⬘-OH group of
`the nucleotides with a reversible moiety allows for the addition
`of all four nucleotides simultaneously in performing SBS,
`thereby increasing accuracy and reducing the number of cycles
`needed. However,
`it is essential that the capping group be
`efficiently removed from the DNA extension products to regen-
`erate the 3⬘-OH group for continuous polymerase reactions. Our
`previous research efforts have firmly established the molecular
`level strategy to rationally modify the nucleotides by capping the
`3⬘-OH with a small chemically reversible moiety for SBS (16–
`20). Building on our successful 3⬘-O-modification strategy for the
`synthesis of the NRTs, we have explored alternative chemically
`reversible groups for capping the 3⬘-OH of the nucleotides. In
`1991, Zavgorodny et al. (25) reported the capping of the 3⬘-OH
`group of the nucleoside with an azidomethyl moiety, which can
`be chemically cleaved under mild condition with triphenylphos-
`phine. Various 3⬘-O-azidomethyl nucleoside analogues have
`subsequently been synthesized (26). Cleavable fluorescent NRTs
`with a variety of 3⬘-O-modification groups, including the azidom-
`ethyl moiety, have been recently proposed for SBS (27), follow-
`ing the general principle that we reported (16, 18, 20) to design
`the NRTs that can be recognized as substrates for DNA poly-
`merase by attaching a cleavable fluorophore to a specific loca-
`tion on the base and capping the 3⬘-OH with a small chemically
`reversible moiety.
`We synthesized and evaluated four 3⬘-O-azidomethyl-
`modified NRTs (3⬘-O-N3-dNTPs) (Fig. 1) for the hybrid SBS.
`The 3⬘-O-modified NRTs containing an azidomethyl group to
`cap the 3⬘-OH on the ribose ring were synthesized based on a
`
`Structures of the nucleotide reversible terminators, 3⬘-O-N3-dATP,
`Fig. 1.
`3⬘-O-N3-dCTP, 3⬘-O-N3-dGTP, and 3⬘-O-N3-dTTP.
`
`method similar to that reported by Zavgorodny et al. (25, 26) as
`described in SI Text. The 3⬘-O-azidomethyl group on the DNA
`extension product generated by incorporating each of the NRTs
`is efficiently removed by the Staudinger reaction by using
`aqueous Tris(2-carboxyethyl) phosphine (TCEP) solution (28,
`29) followed by hydrolysis to yield a free 3⬘-OH group for
`elongating the DNA chain in subsequent cycles of the hybrid SBS
`(Fig. S2 A).
`To demonstrate the feasibility of carrying out the hybrid SBS
`on a DNA chip, we designed and synthesized 4 cleavable
`f luorescent dideoxynucleotides, ddNTP-N3-f luorophores
`(ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-
`ROX, and ddGTP-N3-Cy5) (Fig. 2). The ddNTP-N3-fluoro-
`phores will be combined with the 4 NRTs (Fig. 1) to perform the
`hybrid SBS. Modified DNA polymerases have been shown to be
`highly tolerant to nucleotide modifications with bulky groups at
`the 5 position of pyrimidines (C and U) and the 7 position of
`purines (A and G) (30). Thus, we attached each unique fluoro-
`phore to the 5 position of C/U and the 7 position of A/G through
`a cleavable linker, which is also based on an azido-modified
`moiety (29) as a trigger for cleavage, a mechanism that is similar
`to the removal of the 3⬘-O-azidomethyl group (Fig. S2B). The
`synthesis and characterization of the cleavable fluorescent
`dideoxynucleotides in Fig. 2 are described in SI Text. The
`ddNTP-N3-fluorophores are found to efficiently incorporate
`into the growing DNA strand to terminate DNA synthesis for
`sequence determination. The fluorophore on a DNA extension
`product, which is generated by incorporation of the cleavable
`fluorescent ddNTPs, is removed rapidly and quantitatively by
`TCEP from the DNA extension product in aqueous solution.
`
`Continuous Polymerase Extension by Using 3ⴕ-O-Modified NRTs and
`Characterization by MALDI-TOF Mass Spectrometry. To verify that
`the 3⬘-O-N3-dNTPs incorporate accurately in a base-specific
`manner in the polymerase reaction, four continuous DNA
`extension and cleavage reactions were carried out in solution by
`using 3⬘-O-N3-dNTPs as substrates. This allowed the isolation of
`the DNA product at each step for detailed molecular structure
`characterization as shown in Fig. 3. The first extension product
`5⬘-primer-C-N3-3⬘ (1) was desalted and analyzed by using
`MALDI-TOF MS (Fig. 3A). This product was then incubated in
`aqueous TCEP solution to remove the azidomethyl moiety to
`yield the cleavage product (2) with a free 3⬘-OH group, which
`was also analyzed by using MALDI-TOF MS (Fig. 3B). As can
`be seen from Fig. 3A, the MALDI-TOF MS spectrum consists
`of a distinct peak corresponding to the DNA extension product
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`The polymerase extension scheme (Left) and MALDI-TOF MS spectra
`Fig. 3.
`of the four consecutive extension products and their cleavage products (Right)
`using four 3⬘-O-N3-dNTPs. Primer extended with 3⬘-O-N3-dCTP to yield product
`1 (A), and its cleavage product 2 (B); product 2 extended with 3⬘-O-N3-dGTP to
`yield product 3 (C), and its cleavage product 4 (D); product 4 extended with
`3⬘-O-N3-dATP to yield product 5 (E), and its cleavage product 6 (F); product 6
`extended with 3⬘-O-N3-dTTP to yield product 7 (G), and its cleavage product 8
`(H). The azidomethyl moiety capping the 3⬘-OH of the DNA extension products
`is completely removed by TCEP aqueous solution to continue the polymerase
`reaction.
`
`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 incuba-
`tion of the DNA extension products in an aqueous TCEP
`solution led to the cleavage of the linker tethering the fluoro-
`phore to the dideoxynucleotide. Fig. 4 B, D, F, and H shows the
`cleavage results for the DNA products extended with ddNTP-
`N3-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 3ⴕ-O-Modified NRTs by the Hybrid SBS Ap-
`proach. In our four-color hybrid SBS approach, the identity of the
`incorporated nucleotide is determined by the unique fluores-
`cence 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-N3-
`fluorophores and 3⬘-O-N3-dNTPs during the polymerase reac-
`tion determines how much of the ddNTP-N3-fluorophores in-
`corporate 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-fluoro-
`
`Structures of the cleavable fluorescent ddNTPs: ddCTP-N3-Bodipy-FL-
`Fig. 2.
`510 (␭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).
`
`5⬘-primer-C-N3-3⬘ (1) (m/z 8,310), which confirms that the NRT
`is incorporated base-specifically by DNA polymerase into a
`growing DNA strand. Fig. 3B shows the cleavage result on the
`DNA extension product. The extended DNA mass peak at m/z
`8,310 completely disappeared, whereas the peak corresponding
`to the cleavage product 5⬘-primer-C-3⬘ (2) appears as the sole
`dominant peak at m/z 8,255, which establishes that TCEP
`incubation completely cleaves the 3⬘-O-azidomethyl group with
`high efficiency. The next extension reaction was carried out by
`using this cleaved product, which now has a free 3⬘-OH group,
`as a primer to yield a second extension product, 5⬘-primer-CG-
`N3-3⬘ (3) (m/z 8,639; Fig. 3C). As described above, the extension
`product (3) was cleaved to generate product (4) for further MS
`analysis yielding a single peak at m/z 8,584 (Fig. 3D). The third
`extension reaction to yield 5⬘-primer-CGA-N3-3⬘ (5) (m/z 8,952;
`Fig. 3E), the fourth extension to yield 5⬘-primer-CGAT-N3-3⬘ (7)
`(m/z 9,256; Fig. 3G) and their cleavage to yield products (6) (m/z
`8,897; Fig. 3F) and (8) (m/z 9,201; Fig. 3H) were similarly carried
`out and analyzed by MALDI-TOF MS. These results demon-
`strate that all four 3⬘-O-N3-dNTPs are successfully synthesized
`and efficiently incorporated base-specifically into the growing
`DNA strand in a continuous polymerase reaction as reversible
`terminators and the 3⬘-OH capping group on the DNA extension
`products is quantitatively cleaved by TCEP.
`
`Polymerase Extension by Using Cleavable Fluorescent Dideoxynucle-
`otide Terminators and Characterization by MALDI-TOF Mass Spec-
`trometry. To verify that the four cleavable fluorescent ddNTPs
`(ddCTP-N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-
`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 tem-
`plates whose next complementary base was either A, C, G, or T
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`Guo et al.
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`PNAS 兩
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`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).
`
`phores to produce sufficient fluorescent signals that are above
`the fluorescence detection system’s sensitivity threshold for
`sequence determination. As the sequencing cycle continues, the
`amount of the ddNTP-N3-fluorophores needs to be gradually
`increased to maintain the fluorescence emission strength for
`detection. Following these guidelines, we performed the hybrid
`SBS on a chip-immobilized DNA template by using the 3⬘-O-
`N3-dNTP/ddNTP-N3-fluorophore combination and the results
`are shown in Fig. 5. The general four-color sequencing reaction
`scheme on a DNA chip is shown in Fig. 5A.
`The de novo sequencing reaction on the chip was initiated by
`extending the self-priming DNA by using a solution consisting of
`four 3⬘-O-N3-dNTPs and four ddNTP-N3-fluorophores, and 9oN
`DNA polymerase. The hybrid SBS allows for the addition of all
`eight nucleotide substrates simultaneously to unambiguously
`determine DNA sequences. This reduces the number of steps
`needed to complete the sequencing cycle, while increasing the
`sequencing accuracy because of competition among the sub-
`strates in the polymerase reaction. The DNA products extended
`by ddNTP-N3-fluorophores, after fluorescence detection for
`sequence determination and cleavage, are no longer involved in
`the subsequent polymerase reaction cycles because they are
`permanently terminated. Therefore, further polymerase reac-
`tion only occurs on a DNA strand that incorporates the 3⬘-O-
`N3-dNTPs, which subsequently turn back into natural nucleotide
`on cleavage of the 3⬘-OH capping group, and should have no
`deleterious effect on the polymerase binding to incorporate
`subsequent nucleotides for growing the DNA chains. However,
`successive addition of the previously designed cleavable fluo-
`rescent NRTs (20, 27, 29) into a growing DNA strand during SBS
`leads to a newly synthesized DNA chain with a leftover propargyl
`amino group at each nucleobase. This may interfere with the
`ability of the enzyme to efficiently incorporate the next incoming
`nucleotide, which will lead to loss of synchrony and thereby
`
`reduction in the read length. This challenge might potentially be
`overcome by reengineering DNA polymerases that efficiently
`recognize and accept the modified DNA strand, or by alternative
`design of the fluorescent NRTs (31).
`To negate any lagging fluorescence signal that is caused by a
`previously unextended priming strand, a synchronization step
`was added to reduce the amount of unextended priming strands
`after the initial extension reaction shown in the scheme of Fig.
`5A. A synchronization reaction mixture consisting of just the four
`3⬘-O-N3-dNTPs in relatively high concentration was used along
`with the 9oN DNA polymerase to extend any remaining priming
`strands that retain a free 3⬘-OH group to synchronize the
`incorporation.
`The four-color images from a fluorescence scanner for each
`step of the hybrid SBS on a chip is shown in Fig. 5B. The first
`extension of the primer by the complementary fluorescent
`ddNTP, ddCTP-N3-Bodipy-FL-510, was confirmed by observing
`a blue signal (the emission from Bodipy-FL-510) [Fig. 5B (1)].
`After fluorescent signal detection, the surface was immersed in
`a TCEP solution to cleave both the fluorophore from the DNA
`product extended with ddNTP-N3-fluorophores and the 3⬘-O-
`azidomethyl group from the DNA product extended with 3⬘-O-
`N3-dNTPs. The surface of the chip was then washed, and a
`negligible residual fluorescent signal was detected, confirming
`cleavage of the fluorophore [Fig. 5B (2)]. This was followed by
`another extension reaction with the 3⬘-O-N3-dNTP/ddNTP-N3-
`fluorophore solution to incorporate the next nucleotide com-
`plementary to the subsequent base on the template. The entire
`process of incorporation, synchronization, detection, and cleav-
`age was performed multiple times to identify 32 successive bases
`in the DNA template. The plot of the fluorescence intensity vs.
`the progress of sequencing extension (raw four-color sequencing
`data) is shown in Fig. 5C. The DNA sequences including the
`homopolymer regions are unambiguously identified with no
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`9148 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0804023105
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`SCIENCES
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`APPLIEDBIOLOGICAL
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`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
`Fig. 5.
`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.
`
`errors from the four-color raw fluorescence data without any
`processing. Similar four-color sequencing data were obtained for
`a variety of DNA templates (Fig. S3).
`
`Conclusion
`We have synthesized four 3⬘-O-N3-dNTPs along with four
`cleavable fluorescent ddNTPs and used them to produce four-
`color de novo DNA sequencing data on a chip by the hybrid SBS
`approach that has the following advantages. With the 3⬘-O-N3-
`dNTPs, after cleavage of the 3⬘-OH capping group of the DNA
`extension product, there are no traces of modification left on the
`growing DNA strand. Therefore, there will be no adverse effect
`on the DNA polymerase for the incorporation of the next
`complementary nucleotide. Second, the cleavable fluorescent
`ddNTPs and 3⬘-O-N3-dNTPs are permanent and reversible
`terminators, respectively, which allow the interrogation of each
`base in a serial manner, a key strategy enabling accurate
`determination of homopolymeric regions of DNA. In addition,
`because all of the steps of the nucleotide incorporation, fluo-
`rescence detection for sequence determination, cleavage of the
`
`fluorophore, and the 3⬘-O-azidomethyl group are performed on
`a DNA chip, there is no longer a need for electrophoretic DNA
`fragment separation as in the classical Sanger sequencing
`method.
`We have experimentally determined the ratio of the 3⬘-O-N3-
`dNTPs and ddNTP-N3-fluorophores to yield sequencing read
`length of 32 bases. The signal strength at base 32 is as strong as
`that of the first base (Fig. 5C), indicating it should be possible to
`increase the read length of the hybrid SBS further by optimizing
`the extension conditions to reduce the background fluorescence
`in the later sequencing cycles. The ultimate read length of this
`hybrid SBS system depends on three factors: the number of
`starting DNA molecules on each spot of a DNA chip, the
`reaction efficiency, and the detection sensitivity of the system.
`The read length with the Sanger sequencing method commonly
`reaches ⬎700 bp. The hybrid SBS approach described here may
`have the potential to reach this read length, especially with
`improvements in the sensitivity of the fluorescent detection
`system, where single molecules can be reliably detected.
`With sequencing read length from 14 to 30 bases in the next
`generation DNA sequencing systems, massive parallel digital
`
`Guo et al.
`
`PNAS 兩
`
`July 8, 2008 兩 vol. 105 兩 no. 27 兩 9149
`
`

`

`gene expression analogous to a high-throughput SAGE (32)
`approach has been reported reaching single copy transcript
`sensitivity (33), and CHIP-Seq (21–23) based on sequencing tags
`of ⬇25 bases has led to many new discoveries in genome function
`and regulation. It is well established that millions of different
`PCR templates can be generated on a solid surface through
`emulsion PCR or clonal amplification (23, 34). Thus, future
`implementation of the hybrid SBS approach on a high-density
`bead array platform will provide a high-throughput and accurate
`DNA sequencing system with wide applications in genome
`biology and biomedical research.
`
`Materials and Methods
`Synthesis of 3ⴕ-O-N3-dNTPs and ddNTP-N3-Fluorophores. The synthesis and
`characterization of the 3⬘-O-modified NRTs (3⬘-O-N3-dCTP, 3⬘-O-N3-dTTP, 3⬘-
`O-N3-dATP, and 3⬘-O-N3-dGTP) and the cleavable fluorescent ddNTPs (ddCTP-
`N3-Bodipy-FL-510, ddUTP-N3-R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) are de-
`scribed in SI Text.
`
`Continuous Polymerase Extension by Using 3ⴕ-O-Modified NRTs in Solution and
`Characterization by MALDI-TOF MS. We characterized the above four 3⬘-O-
`modified NRTs by performing four continuous DNA-extension reactions se-
`quentially by using a self-priming DNA template. The experimental proce-
`dures are described in SI Text.
`
`Polymerase Extension by Using Cleavable Fluorescent Dideoxynucleotide Ter-
`minators in Solution and Characterization by MALDI-TOF MS. We characterized
`the four cleavable fluorescent ddNTPs (ddCTP-N3-Bodipy-FL-510, ddUTP-N3-
`R6G, ddATP-N3-ROX, and ddGTP-N3-Cy5) by performing four separate DNA-
`extension reactions, each with a different self-priming DNA template allow-
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`ing the four ddNTP analogues to be incorporated. The experimental
`procedures are described in SI Text.
`
`Four-Color DNA Sequencing on a Chip by Using Cleavable Fluorescent
`Dideoxynucleotides and 3ⴕ-O-modified NRTs by the Hybrid SBS Approach. The
`DNA template sequence and the immobilization procedure to construct the
`DNA chip are described in SI Text. Ten microliters of a solution consisting of
`ddCTP-N3-Bodipy-FL-510 (10 fmol), ddUTP-N3-R6G (20 fmol), ddATP-N3-ROX
`(40 fmol), ddGTP-N3-Cy5 (20 fmol), 3⬘-O-N3-dCTP (22 pmol), 3⬘-O-N3-dTTP (22
`pmol), 3⬘-O-N3-dATP (22 pmol), 3⬘-O-N3-dGTP (4 pmol), 1 unit of 9oN DNA
`polymerase(exo-) A485L/Y409V, 20 nmol of MnCl2 and 1⫻ Thermopol II reac-
`tion buffer was spotted on the DNA chip. The nucleotide complementary to
`the DNA template was allowed to incorporate into the primer at 65°C for 15
`min. To synchronize any unextended templates, an extension solution con-
`sisting of 38 pmol each of 3⬘-O-N3-dTTP, 3⬘-O-N3-dATP, 3⬘-O-N3-dGTP, and 75
`pmol of 3⬘-O-N3-dCTP, 1 unit of 9oN DNA polymerase(exo-) A485L/Y409V, 20
`nmol of MnCl2 and 1⫻ Thermopol II reaction buffer was added to the same
`spot and incubated at 65°C for 15 min. After washing with SPSC buffer
`containing 0.1% Tween-20 for 1 min, the chip was rinsed with dH2O, and then
`scanned with a four-color fluorescence scanner as described in ref. 20. To
`perform the cleavage, the DNA chip was placed inside a cham