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`Four-color DNA sequencing by synthesis using
`cleavable fluorescent nucleotide
`reversible terminators
`
`Jingyue Ju*†‡, Dae Hyun Kim*§, Lanrong Bi*†, Qinglin Meng*†¶, Xiaopeng Bai*†¶, Zengmin Li*†, Xiaoxu Li*†,
`Mong Sano Marma*†, Shundi Shi*, Jian Wu*†¶, John R. Edwards*†, Aireen Romu*, and Nicholas J. Turro†‡¶
`
`*Columbia Genome Center, Columbia University College of Physicians and Surgeons, New York, NY 10032; and Departments of †Chemical
`Engineering, ¶Chemistry, and §Biomedical Engineering, Columbia University, New York, NY 10027
`
`Contributed by Nicholas J. Turro, October 26, 2006 (sent for review October 1, 2006)
`
`DNA sequencing by synthesis (SBS) on a solid surface during
`polymerase reaction offers a paradigm to decipher DNA sequences.
`We report here the construction of such a DNA sequencing system
`using molecular engineering approaches. In this approach, four
`nucleotides (A, C, G, T) are modified as reversible terminators by
`attaching a cleavable fluorophore to the base and capping the
`3ⴕ-OH group with a small chemically reversible moiety so that they
`are still recognized by DNA polymerase as substrates. We found
`that an allyl moiety can be used successfully as a linker to tether a
`fluorophore to 3ⴕ-O-allyl-modified nucleotides, forming chemically
`cleavable fluorescent nucleotide reversible terminators, 3ⴕ-O-allyl-
`dNTPs-allyl-fluorophore, for application in SBS. The fluorophore
`and the 3ⴕ-O-allyl group on a DNA extension product, which is
`generated by incorporating 3ⴕ-O-allyl-dNTPs-allyl-fluorophore in a
`polymerase reaction, are removed simultaneously in 30 s by Pd-
`catalyzed deallylation in aqueous buffer solution. This one-step
`dual-deallylation reaction thus allows the reinitiation of the poly-
`merase reaction and increases the SBS efficiency. DNA templates
`consisting of homopolymer regions were accurately sequenced by
`using this class of fluorescent nucleotide analogues on a DNA chip
`and a four-color fluorescent scanner.
`
`DNA chip
`
`DNA sequencing is driving genomics research and discovery.
`
`The completion of the Human Genome Project has set the
`stage for screening genetic mutations to identify disease genes on
`a genome-wide scale (1). Accurate high-throughput DNA sequenc-
`ing methods are needed to explore the complete human genome
`sequence for applications in clinical medicine and health care. To
`overcome the limitations of the current electrophoresis-based
`sequencing technology (2–5), a variety of new DNA-sequencing
`methods have been investigated. Such approaches include sequenc-
`ing by hybridization (6), mass spectrometry-based sequencing (7–
`9), sequence-specific detection of single-stranded DNA using en-
`gineered nanopores (10), and sequencing by ligation (11). More
`recently, DNA sequencing by synthesis (SBS) approaches such as
`pyrosequencing (12), sequencing of single DNA molecules (13),
`and polymerase colonies (14) have been widely explored.
`The concept of DNA SBS was revealed in 1988 with an attempt
`to sequence DNA by detecting the pyrophosphate group that is
`generated when a nucleotide is incorporated in a DNA polymerase
`reaction (15). Pyrosequencing, which was developed based on this
`concept and an enzymatic cascade, has been explored for genome
`sequencing (16). However, there are inherent difficulties in this
`method for determining the number of incorporated nucleotides in
`homopolymeric regions of the template. Additionally, each of the
`four nucleotides needs to be added and detected separately, which
`increases the overall detection time. The accumulation of unde-
`graded nucleotides and other components could also lower the
`accuracy of the method when sequencing a long DNA template. It
`is thus desirable to have a simple method to directly detect a
`reporter group attached to the nucleotide that is incorporated into
`
`a growing DNA strand in the polymerase reaction rather than
`relying on a complex enzymatic cascade. The SBS scheme based on
`fluorescence detection coupled with a chip format has the potential
`to markedly increase the throughput of DNA sequencing projects.
`Consequently, several groups have investigated such a system with
`an aim to construct an ultra high-throughput DNA sequencing
`method (17, 18). Thus far, no complete success of using such a
`system to unambiguously sequence DNA has been published.
`Previous work in the literature exploring the SBS method is
`mostly focused on designing and synthesizing a cleavable chemical
`moiety that is linked to a fluorescent dye to cap the 3⬘-OH group
`of the nucleotides (19–21). The rationale is that, after the fluoro-
`phore is removed, the 3⬘-OH would be regenerated to allow
`subsequent nucleotide addition. However, no success has been
`reported for the incorporation of such a nucleotide with a cleavable
`fluorescent dye on the 3⬘ position by DNA polymerase into a
`growing DNA strand. The reason is that the 3⬘ position on the
`deoxyribose is very close to the amino acid residues in the active site
`of the polymerase, and the polymerase is therefore sensitive to
`modification in this area of the ribose ring, especially with a large
`fluorophore (22).
`It is known that some modified DNA polymerases are highly
`tolerable for nucleotides with extensive modifications with bulky
`groups such as energy transfer dyes at the 5-position of the
`pyrimidines (T and C) and 7-position of purines (G and A) (23, 24).
`The ternary complexes of a rat DNA polymerase, a DNA template-
`primer, and dideoxycytidine triphosphate have been determined
`(22), which supports this fact. We thus reasoned that if a unique
`fluorescent dye is linked to the 5-position of the pyrimidines (T and
`C) and the 7-position of purines (G and A) via a cleavable linker,
`and a small chemical moiety is used to cap the 3⬘-OH group, the
`resulting nucleotide analogues should be able to incorporate into
`the growing DNA strand as terminators. Based on this rationale, we
`proposed an SBS approach using cleavable fluorescent nucleotide
`analogues as reversible terminators to sequence surface-
`immobilized DNA [supporting information (SI) Fig. 6] (25). In this
`approach, the nucleotides are modified at two specific locations so
`that they are still recognized by DNA polymerase as substrates: (i)
`a different fluorophore with a distinct fluorescent emission is linked
`to each of the four bases through a cleavable linker and (ii) the
`
`Author contributions: J.J. and N.J.T. designed research; D.H.K., L.B., Q.M., X.B., Z.L., X.L., M.S.M.,
`S.S., J.W., J.R.E., and A.R. performed research; and J.J., D.H.K., and Q.M. wrote the paper.
`
`The authors declare no conflict of interest.
`
`Freely available online through the PNAS open access option.
`
`Abbreviations: SBS, sequencing by synthesis; bodipy, 4,4-difluoro-4-bora-3␣,4␣-diaza-s-
`indacene; ROX, 6-carboxy-X-rhodamine; R6G, 6-carboxyrhodamine-6G.
`‡To whom correspondence may be addressed at: Room 405A, Russ Berrie Medical Science
`Pavilion, Columbia Genome Center, Columbia University College of Physicians and Sur-
`geons, New York, NY 10032. E-mail: ju@c2b2.columbia.edu or njt3@columbia.edu.
`
`This article contains supporting information online at www.pnas.org/cgi/content/full/
`0609513103/DC1.
`
`© 2006 by The National Academy of Sciences of the USA
`
`www.pnas.org兾cgi兾doi兾10.1073兾pnas.0609513103
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`3⬘-OH group is capped by a small chemically reversible moiety.
`DNA polymerase incorporates only a single nucleotide analogue
`complementary to the base on a DNA template covalently linked
`to a surface. After incorporation, the unique fluorescence emission
`is detected to identify the incorporated nucleotide and the fluoro-
`phore is subsequently removed. The 3⬘-OH group is then chemically
`regenerated, which allows the next cycle of the polymerase reaction
`to proceed. Because the large surface on a DNA chip can have a
`high density of different DNA templates spotted, each cycle can
`identify many bases in parallel, allowing the simultaneous sequenc-
`ing of a large number of DNA molecules. We have previously
`established the feasibility of performing SBS on a chip using four
`photocleavable fluorescent nucleotide analogues (26) and discov-
`ered that an allyl group can be used as a cleavable linker to bridge
`a fluorophore to a nucleotide (27). We have also reported the
`design and synthesis of two photocleavable fluorescent nucleotides
`as reversible terminators for polymerase reaction (28, 29).
`Our previous research efforts have firmly established the
`molecular level strategy to rationally modify the nucleotides by
`attaching a cleavable fluorescent dye to the base and capping the
`3⬘-OH with a small chemically reversible moiety for SBS. This
`approach was recently adopted by Genomics Industry to poten-
`tially provide a platform for DNA sequencing (30). Here we
`report the design and synthesis of four chemically cleavable
`fluorescent nucleotide analogues as reversible terminators for
`SBS. Each of the nucleotide analogues contains a 3⬘-O-allyl
`group and a unique fluorophore with a distinct fluorescence
`emission at the base through a cleavable allyl linker. We first
`established that these nucleotide analogues are good substrates
`for DNA polymerase in a solution-phase DNA extension reac-
`tion and that the fluorophore and the 3⬘-O-allyl group can be
`removed with high efficiency in aqueous solution. We then
`performed SBS using these four chemically cleavable fluorescent
`nucleotide analogues as reversible terminators to identify ⬇20
`continuous bases of a DNA template immobilized on a chip.
`Accurate DNA sequences were obtained for DNA templates
`containing homopolymer sequences. The DNA template was
`immobilized on the surface of the chip that contains a PEG
`linker with 1,3-dipolar azide-alkyne cycloaddition chemistry.
`These results indicated that successful cleavable fluorescent
`nucleotide reversible terminators for four-color DNA sequenc-
`ing by synthesis can be designed by attaching a cleavable
`fluorophore to the base and capping the 3⬘-OH with a small
`chemically reversible moiety so that they are still recognized by
`DNA polymerase as substrates. Further optimization of the
`approach will lead to even longer sequencing read lengths.
`
`Results and Discussion
`Design and Synthesis of Chemically Cleavable Fluorescent Nucleotide
`Analogues as Reversible Terminators for SBS. To demonstrate the
`feasibility of carrying out de novo DNA sequencing by synthesis on
`a chip, four chemically cleavable fluorescent nucleotide analogues
`(3⬘-O-allyl-dCTP-allyl-bodipy-FL-510, 3⬘-O-allyl-dUTP-allyl-R6G,
`3⬘-O-allyl-dATP-allyl-ROX and 3⬘-O-allyl-dGTP-allyl-bodipy-650/
`Cy5) (R6G, 6-carboxyrhodamine-6G; Rox, 6-carboxy-X-rhoda-
`mine; bodipy, 4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene; Fig. 1)
`were designed and synthesized as reversible terminators for DNA
`polymerase reaction. 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). Thus, we attached each unique fluorophore to
`the 5-position of C/U and the 7 position of A/G through an allyl
`carbamate linker. However, due to the close proximity of the 3⬘
`position on the sugar ring of a nucleotide to the amino acid residues
`of the active site of the DNA polymerase, a relatively small allyl
`moiety was chosen as the 3⬘-OH reversible capping group. We have
`found that the fluorophore and the 3⬘-O-allyl group on a DNA
`extension product, which is generated by incorporation of the
`
`Structures of 3⬘-O-allyl-dCTP-allyl-bodipy-FL-510 [␭abs(max) ⫽ 502 nm;
`Fig. 1.
`␭em(max) ⫽ 510 nm], 3⬘-O-allyl-dUTP-allyl-R6G [␭abs(max) ⫽ 525 nm; ␭em(max) ⫽ 550
`nm], 3⬘-O-allyl-dATP-allyl-ROX [␭abs(max) ⫽ 585 nm; ␭em(max) ⫽ 602 nm], and
`3⬘-O-allyl-dGTP-allyl-bodipy-650 [␭abs(max) ⫽ 630 nm; ␭em(max) ⫽ 650 nm].
`
`chemically cleavable fluorescent nucleotide analogues, are removed
`simultaneously in 30 s by Pd-catalyzed deallylation in aqueous
`solution. This one-step dual-deallylation reaction thus allows the
`re-initiation of the polymerase reaction. The detailed synthesis
`procedure and characterization of the four nucleotide analogues in
`Fig. 1 are described in SI Text.
`To verify that these fluorescent nucleotide analogues are incor-
`porated accurately in a base-specific manner in a polymerase
`reaction, four continuous steps of DNA extension and deallylation
`were carried out in solution. This allows the isolation of the DNA
`product at each step for detailed molecular structure characteriza-
`tion by MALDI-TOF mass spectrometry (MS) as shown in Fig. 2.
`The first extension product 5⬘-U(allyl-R6G)-3⬘-O-allyl (1) was
`purified by HPLC and analyzed using MALDI-TOF MS (Fig. 2A).
`This product was then deallylated using a Pd-catalyzed deallylation
`mixture [1⫻ Thermopol I reaction buffer/Na2PdCl4/P(PhSO3Na)3].
`The active Pd catalyst is generated from Na2PdCl4 and a ligand
`P(PhSO3Na)3 (TPPTS) to mediate the deallylation reaction in
`DNA compatible aqueous condition to simultaneously cleave both
`the fluorophore and the 3⬘-O-allyl group (28). The deallylated
`product (2) was also analyzed by using MALDI-TOF MS (Fig. 2B).
`As can be seen in Fig. 2A, the MALDI-TOF MS spectrum consists
`of a distinct peak at m/z 6469 corresponding to the DNA extension
`product 5⬘-U(allyl-R6G)-3⬘-O-allyl (1), which confirms that the
`nucleotide analogue can be incorporated base specifically among
`pool of all four (A, C, G, T) by DNA polymerase into a growing
`DNA strand. Fig. 2B shows the deallylation result of the above
`DNA product. The peak at m/z 6469 has completely disappeared,
`whereas the peak corresponding to the dual deallylated product
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`The polymerase extension scheme (Left) and MALDI-TOF MS spectra of the four consecutive extension products and their deallylated products (Right).
`Fig. 2.
`Primer extended with 3⬘-O-allyl-dUTP-allyl-R6G (1), and its deallylated product 2; Product 2 extended with 3⬘-O-allyl-dGTP-allyl-bodipy-650 (3), and its deallylated
`product 4; Product 4 extended with 3⬘-O-allyl-dATP-allyl-ROX (5), and its deallylated product 6; Product 6 extended with 3⬘-O-allyl-dCTP-allyl-bodipy-FL-510 (7),
`and its deallylated product (8). After 30 s of incubation with the palladium/TPPTS mixture at 70°C, deallylation is complete with both the fluorophores and the
`3⬘-O-allyl groups cleaved from the extended DNA products.
`
`5⬘–U (2) appears as the sole dominant peak at m/z 5870, which
`establishes that the Pd-catalyzed deallylation reaction completely
`cleaves both the fluorophore and the 3⬘-O-allyl group with high
`efficiency without damaging the DNA. The next extension reaction
`was carried out by using this deallylated DNA product with a free
`3⬘-OH group regenerated as a primer along with four allyl modified
`fluorescent nucleotide mixture to yield an extension product 5⬘-
`UG(allyl-bodipy-650)-3⬘-O-allyl (3). As described above, the exten-
`sion product 3 was analyzed by MALDI-TOF MS producing a
`dominant peak at m/z 6984 (Fig. 2C), and then deallylated for
`further MS analysis yielding a single peak at m/z 6256 (product 4)
`(Fig. 2D). The third extension reaction yielding 5⬘-UGA(allyl-
`ROX)-3⬘-O-allyl (5), the fourth extension reaction yielding 5⬘-
`UGAC(allyl-bodipy-FL-510)-3⬘-O-allyl (7), and their deallylation
`reactions to yield products 6 and 8 were similarly carried out and
`analyzed by MALDI-TOF MS as shown in Fig. 2 E–H. The
`chemical structures of the extension and cleavage products for each
`step are shown in SI Fig. 7. These results demonstrate that the
`above-synthesized four chemically cleavable fluorescent nucleotide
`analogues are successfully incorporated with high fidelity into the
`growing DNA strand in a polymerase reaction, and furthermore,
`both the fluorophore and the 3⬘-O-allyl group are efficiently
`removed by using a Pd-catalyzed deallylation reaction, which makes
`it feasible to use them for SBS on a chip.
`
`Four-Color DNA Sequencing with Chemically Cleavable Fluorescent
`Nucleotide Analogues as Reversible Terminators on a DNA Chip. The
`chemically cleavable fluorescent nucleotide analogues were then
`
`used in an SBS reaction to identify the sequence of the DNA
`template immobilized on a solid surface. A site-specific 1,3-dipolar
`cycloaddition coupling chemistry was used to covalently immobilize
`the alkyne-labeled self-priming DNA template on the azido-
`functionalized surface in the presence of a Cu(I) catalyst. The
`principal advantage offered by the use of a self-priming moiety as
`compared with using separate primers and templates is that the
`covalent linkage of the primer to the template in the self-priming
`moiety prevents any possible dissociation of the primer from the
`template during the process of SBS. To prevent nonspecific ab-
`sorption of the unincorporated fluorescent nucleotides on the
`surface of the chip, a PEG linker is introduced between the DNA
`templates and the chip surface (SI Fig. 8). This approach was shown
`to produce very low background fluorescence after cleavage to
`remove the fluorophore as demonstrated by the DNA sequencing
`data described below.
`We first performed SBS on a chip-immobilized DNA template
`that has no homopolymer sequences using the four chemically
`cleavable fluorescent nucleotide reversible terminators (3⬘-O-allyl-
`dCTP-allyl-bodipy-FL-510, 3⬘-O-allyl-dUTP-allyl-R6G, 3⬘-O-allyl-
`dATP-allyl-ROX and 3⬘-O-allyl-dGTP-allyl-Cy5), and the results
`are shown in Fig. 3. The structure of the self-priming DNA moiety
`is shown schematically in Fig. 3A, with the first 13 nucleotide
`sequences immediately after the priming site. The de novo sequenc-
`ing reaction on the chip was initiated by extending the self-priming
`DNA using a solution containing all four 3⬘-O-allyl-dNTPs-allyl-
`fluorophore, and a 9°N mutant DNA polymerase. To negate any
`lagging fluorescence signal that is caused by previously unextended
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`Four-color sequencing by synthesis data on a DNA chip. (A) Reaction scheme of SBS on a chip using four chemically cleavable fluorescent nucleotides.
`Fig. 3.
`(B) The scanned four-color fluorescence images for each step of SBS on a chip: (1) incorporation of 3⬘-O-allyl-dGTP-allyl-Cy5; (2) cleavage of allyl-Cy5 and 3⬘-allyl
`group; (3) incorporation of 3⬘-O-allyl-dATP-allyl-ROX; (4) cleavage of allyl-ROX and 3⬘-allyl group; (5) incorporation of 3⬘-O-allyl-dUTP-allyl-R6G; (6) cleavage of
`allyl-R6G and 3⬘-allyl group; (7) incorporation of 3⬘-O-allyl-dCTP-allyl-bodipy-FL-510; (8) cleavage of allyl-bodipy-FL-510 and 3⬘-allyl group; images 9 –25 are
`similarly produced. (C) A plot (four-color sequencing data) of raw fluorescence emission intensity at the four designated emission wavelength of the four
`chemically cleavable fluorescent nucleotides vs. the progress of sequencing extension.
`
`priming strand, a synchronization step was added to reduce the
`amount of unextended priming strands after the extension with the
`fluorescent nucleotides. A synchronization reaction mixture con-
`sisting of all four 3⬘-O-allyl-dNTPs (Fig. 4), which have a higher
`polymerase incorporation efficiency due to the lack of a fluoro-
`phore compared with the bulkier 3⬘-O-allyl-dNTPs-allyl-
`fluorophore, was used along with the 9°N mutant DNA polymerase
`to extend any remaining priming strand that has a free 3⬘-OH group
`to synchronize the incorporation. The extension by 3⬘-O-allyl-
`dNTPs also enhances the enzymatic incorporation of the next
`nucleotide analogue, because after cleavage to remove the 3⬘-O-
`allyl group, the DNA product extended by 3⬘-O-allyl-dNTPs carry
`no modification groups. After washing, the extension of the primer
`by only the complementary fluorescent nucleotide was confirmed
`by observing a red signal (the emission from Cy5) in a four-color
`fluorescent scanner (Fig. 3B1). After detection of the fluorescent
`signal, the chip surface was immersed in a deallylation mixture [1⫻
`Thermolpol I reaction buffer/Na2PdCl4/P(PhSO3Na)3] and incu-
`bated for 5 min at 60°C to cleave both the fluorophore and
`3⬘-O-allyl group simultaneously. The chip was then immediately
`immersed in a 3 M Tris䡠HCl buffer (pH 8.5) and incubated for 5 min
`at 60°C to remove the Pd complex. The surface was then washed,
`and a negligible residual fluorescent signal was detected to confirm
`
`cleavage of the fluorophore. This was followed by another extension
`reaction using 3⬘-O-allyl-dNTPs-allyl-fluorophore mixture to incor-
`porate the next fluorescent nucleotide complementary to the
`
`Structures of 3⬘-O-allyl-dATP, 3⬘-O-allyl-dCTP, 3⬘-O-allyl-dGTP, and
`Fig. 4.
`3⬘-O-allyl-dTTP.
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`Fig. 5. Comparison of four-color sequencing by synthesis and pyrosequencing data. (A) Four-color DNA sequencing raw data with our sequencing by synthesis
`chemistry using a template containing two homopolymeric regions. The individual base (A, T, C, G), the 10 repeated A’s, and the five repeated A’s are clearly
`identified. The small groups of peaks between the identified bases are fluorescent background from the DNA chip, which does not build up as the cycle continues.
`(B) The pyrosequencing data of the same DNA template containing the homopolymeric regions (10 T’s and five T’s). The first four individual bases are clearly
`identified. The two homopolymeric regions (10 A’s) and (five A’s) produce two large peaks, which are very difficult to identify the exact sequence from the data.
`
`subsequent base on the template. The entire process of incorpo-
`ration, synchronization, detection, and cleavage was performed
`multiple times using the four chemically cleavable fluorescent
`nucleotide reversible terminators to identify 13 successive bases in
`the DNA template. The fluorescence image of the chip for each
`nucleotide addition is shown in Fig. 3B, and a plot of the fluores-
`cence intensity vs. the progress of sequencing extension (raw
`four-color sequencing data) is shown in Fig. 3C. The DNA se-
`quences are unambiguously identified from the four-color raw
`fluorescence data without any processing.
`
`Comparison of Four-Color SBS with Pyrosequencing. To further verify
`the advantage of SBS method using the four chemically cleavable
`fluorescent nucleotide reversible terminators, we carried out sim-
`ilar sequencing reaction as described above on a DNA template
`which contained two separate homopolymeric regions (stretch of 10
`T’s and five T’s) as shown in Fig. 5A. These sequencing raw data
`were produced by adding all four fluorescent nucleotide reversible
`terminators together to the DNA template immobilized on the chip
`followed by synchronization reaction with four 3⬘-O-allyl-dNTPs,
`detecting the unique fluorescence emission for sequence determi-
`nation, then cleaving the fluorophore and the 3⬘-O-allyl group in
`one step to continue the sequencing cycles. All 20 bases including
`the individual base (A, T, C, G), the 10 repeated A’s and the five
`repeated A’s are clearly identified. The small groups of peaks
`between the identified bases are fluorescent background from the
`DNA chip, which does not build up as the cycle continues. Fig. 5B
`shows the pyrosequencing data of the same DNA template con-
`taining the homopolymeric sequences. The first four individual
`bases are clearly identified. The two homopolymeric regions (10
`
`A’s) and (five A’s) produce two large peaks, but it is very difficult
`to identify the exact sequence from the data.
`
`Conclusion
`We have synthesized and characterized four chemically cleavable
`fluorescent nucleotide analogues and used them to produce four-
`color de novo DNA sequencing data on a chip. In doing so, we have
`achieved two critical requirements for using SBS method to se-
`quence DNA unambiguously. First, a strategy to use a chemically
`reversible moiety to cap the 3⬘-OH group of the nucleotide has been
`successfully implemented so that the nucleotide terminates the
`polymerase reaction to allow the identification of the incorporated
`nucleotide. In addition, these reversible terminators allow for the
`addition of all four nucleotides simultaneously in performing SBS.
`This ultimately reduces the number of cycles needed to complete
`the sequencing cycle, increases sequencing accuracy due to com-
`petition of the four nucleotides in the polymerase reaction, and
`enables accurate determination of homopolymeric regions of DNA
`template. Second, efficient removal of both the fluorophore and the
`3⬘-OH capping group after the fluorescence signal detection have
`successfully been carried out, which increases the overall efficiency
`of SBS.
`The key factor governing the sequencing read length of our
`four-color SBS approach is the step-wise yield that is determined by
`the nucleotide incorporation efficiency and the yield of the cleavage
`of the fluorophore and the 3⬘-OH capping group from the DNA
`extension products. This stepwise yield is ⬇99% based on mea-
`surement of the DNA products in solution phase. The yield on the
`surface is difficult to measure precisely due to fluctuations in the
`fluorescence imaging using the current manual fluorescent scanner.
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`The strong fluorescence signal even for the 20th base shown in Fig.
`5 indicates that we should be able to extend the read length even
`further. In terms of read length, Sanger sequencing is still the gold
`standard with read length of ⬎800 bp but limited in throughput and
`cost. The read length of pyrosequencing is ⬇100 bp, but with high
`error rate due to difficulty in accurately determining the sequences
`of homopolymers. Our four-color SBS read length on a manual
`fluorescent scanner is currently at ⬇20 bp with high accuracy. This
`read length is expected to increase when all of the extension,
`cleavage, and washing steps are automated. The DNA polymerases
`and fluorescent labeling used in the automated four-color Sanger
`sequencing method have undergone almost two decades of consis-
`tent incremental improvements after the basic fluorescent Sanger
`methods were established (2, 3). Following the same route, it is
`expected that the basic principle and strategy outlined in our
`four-color SBS method will stimulate further improvement of the
`sequencing by synthesis methodology with engineering of high-
`performance polymerases tailored for the cleavable fluorescent
`nucleotide terminators and testing alternative linkers and 3⬘-OH
`reversible capping moiety. It has been well established that, by using
`emulsion PCR on microbeads, millions of different DNA templates
`are immobilized on a surface of a chip (11, 16). These high-density
`DNA templates, coupled with our four-color SBS approach, will
`generate a high-throughput (⬎20 million bases per chip) and highly
`accurate platform for a variety of sequencing and digital gene
`expression analysis projects.
`
`Materials and Methods
`Synthesis of 3ⴕ-O-allyl-dNTPs-allyl-Fluorophore. The detailed syn-
`thesis of the chemically cleavable fluorescent nucleotides 3⬘-O-
`allyl-dCTP-allyl-bodipy-FL-510, 3⬘-O-allyl-dUTP-allyl-R6G, 3⬘-
`O-allyl-dATP-allyl-ROX, 3⬘-O-allyl-dGTP-allyl-bodipy-650, and
`3⬘-O-allyl-dGTP-allyl-Cy5 are described in SI Text.
`
`Construction of a Chip with Immobilized Self-Priming DNA Template.
`The DNA chip was constructed as shown in SI Fig. 8, and the
`detailed steps are described in SI Text.
`
`Continuous DNA Polymerase Reaction Using Four Chemically Cleav-
`able Fluorescent Nucleotides as Reversible Terminators in Solution.
`We characterized the four nucleotide analogues 3⬘-O-allyl-dCTP-
`allyl-bodipy-FL-510, 3⬘-O-allyl-dUTP-allyl-R6G, 3⬘-O-allyl-dATP-
`allyl-ROX and 3⬘-O-allyl-dGTP-allyl-bodipy-650, by performing
`four continuous DNA-extension reactions sequentially using a
`primer (5⬘-AGAGGATCCAACCGAGAC-3⬘) and a synthetic
`
`DNA template (5⬘-GTGTACATCAACATCACCTACCACCAT-
`GTCAGTCTCG-GTTGGATCCTCTATTGTGTCCGG-3⬘)
`based on a portion of exon 7 of the human p53 gene. The detailed
`experimental procedures are described in SI Text.
`
`Four-Color SBS Reaction on a Chip with Four Chemically Cleavable
`Fluorescent Nucleotides as Reversible Terminators. Ten microliters
`of a solution consisting of 3⬘-O-allyl-dCTP-allyl-bodipy-FL-510
`(3 pmol), 3⬘-O-allyl-dUTP-allyl-R6G (10 pmol), 3⬘-O-allyl-
`dATP-allyl-ROX (5 pmol), and 3⬘-O-allyl-dGTP-allyl-Cy5 (2
`pmol), 1 unit of 9°N mutant DNA polymerase, and 1⫻ Ther-
`molpol II reaction buffer was spotted on the surface of the chip,
`where the self-primed DNA moiety was immobilized. The
`nucleotide analogue complementary to the DNA template was
`allowed to incorporate into the primer at 68°C for 10 min. To
`synchronize any unincorporated templates, an extension solu-
`tion consisting of 30 pmol each of 3⬘-O-allyl-dCTP, 3⬘-O-allyl-
`dTTP, 3⬘-O-allyl-dATP and 3⬘-O-allyl-dGTP, 1 unit of 9°N
`mutant DNA polymerase, and 1X Thermolpol II reaction buffer
`was spotted on the same spot and incubated at 68°C for 10 min.
`After washing the chip with a SPSC buffer containing 0.1%
`Tween 20 for 5 min, the surface was rinsed with dH2O, dried
`briefly and then scanned with a four-color ScanArray Express
`scanner (PerkinElmer Life Sciences, Boston, MA) to detect the
`fluorescence signal. The four-color scanner is equipped with
`four lasers with excitation wavelengths of 488, 543, 594, and 633
`nm and emission filters centered at 522, 570, 614, and 670 nm.
`For deallylation, the chip was immersed in a deallylation mixture
`[1⫻ Thermolpol I reaction buffer/Na2PdCl4/P(PhSO3Na)3] and
`incubated for 5 min at 60°C. The chip was then immediately
`immersed in 3 M Tris䡠HCl buffer (pH 8.5) and incubated for 5
`min at 60°C. Finally, the chip was rinsed with acetonitrile/dH2O
`(1:1 vol/vol) and dH2O. The chip surface was scanned again to
`compare the intensity of fluorescence after deallylation with the
`original fluorescence intensity. This process was followed by the
`next polymerase extension reaction using 3⬘-O-allyl-dNTPs-allyl-
`fluorophore and 3⬘-O-allyl-dNTPs, with the subsequent washing,
`fluorescence detection, and deallylation processes performed as
`described above. The same cycle was repeated multiple times
`using the four chemically cleavable fluorescent nucleotide mix-
`ture in polymerase extension reaction to obtain de novo DNA
`sequencing data on various different DNA templates.
`
`This work was supported by National Institutes of Health Grants P50
`HG002806 and R01 HG003582 and the Packard Fellowship for Science
`and Engineering.
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