"Click chemistry" 1,3-dipolar cycloaddition between alkynyl 6-carboxyfluorescein (FAM) and azido-labeled single-stranded (ss) DNA was carried out under aqueous conditions to produce FAM-labeled ssDNA in quantitative yield. The FAM-labeled ssDNA was successfully used as a primer to produce DNA sequencing products with single-base resolution in a capillary electrophoresis DNA sequencer with laser-induced fluorescence detection.

DNA sequencing by synthesis on a solid surface offers new paradigms to overcome limitations of electrophoresis-based sequencing methods. Here we report DNA sequencing by synthesis using photocleavable (PC) fluorescent nucleotides [dUTP-PC-4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene (Bodipy)-FL-510, dCTP-PC-Bodipy-650, and dUTP-PC-6-carboxy-X-rhodamine (ROX)] on a glass chip constructed by 1,3-dipolar azide-alkyne cycloaddition coupling chemistry. Each nucleotide analogue consists of a different fluorophore attached to the base through a PC 2-nitrobenzyl linker. We constructed a DNA microarray by using the 1,3-dipolar cycloaddition chemistry to sitespecifically attach azido-modified DNA onto an alkyne-functionalized glass chip at room temperature under aqueous conditions. After verifying that the polymerase reaction could be carried out successfully on the above-described DNA array, we then performed a sequencing reaction on the chip by using a self-primed DNA template. In the first step, we extended the primer using DNA polymerase and dUTP-PC-Bodipy-FL-510, detected the fluorescent signal from the fluorophore Bodipy-FL-510, and then cleaved the fluorophore using 340 nm UV irradiation. This process was followed by extension of the primer with dCTP-PC-Bodipy-650 and the subsequent detection of the fluorescent signal from Bodipy-650 and its photocleavage. The same procedure was also performed by using dUTP-PC-ROX. The entire process was repeated five times by using the three fluorescent nucleotides to identify 7 bases in the DNA template. These results demonstrate that the PC nucleotide analogues can be incorporated accurately into a growing DNA strand during polymerase reaction on a chip, and the fluorophore can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing the continuous identification of the template sequence.1,3-dipolar azide-alkyne cycloaddition ͉ DNA chip ͉ DNA sequencing by synthesis ͉ 2-nitrobenzyl linker ͉ photocleavage T he 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 methods for resequencing the intron͞exon regions of candidate genes are needed to explore the complete human genome sequence for disease-gene discovery. Recent studies also have demonstrated that an important route for identifying functional elements in the human genome is to sequence the genomes of many species that represent a wide sampling of the evolutionary tree (2). To overcome the limitations of the current sequencing technology based on electrophoresis using laser-induced fluorescence detection (3-5), a variety of new DNA-sequencing methods have been explored. Such techniques include pyrosequencing (6), MS sequencing (7-9), sequencing by hybridization (10), sequence-specific detection of singlestranded DNA using engineered nanopores (11), and sequencing of single DNA molecules (12) and polymerase colonies (13).Recently, new DNA-sequencing approaches based on sequencing by syn...

; R6G, 6-carboxyrhodamine-6G). Each nucleotide analogue consists of a different fluorophore attached to the 5 position of the pyrimidines and the 7 position of the purines through a photocleavable 2-nitrobenzyl linker. After verifying that these nucleotides could be successfully incorporated into a growing DNA strand in a solutionphase polymerase reaction and the fluorophore could be cleaved using laser irradiation (Ϸ355 nm) in 10 sec, we then performed an SBS reaction on a chip that contains a self-priming DNA template covalently immobilized by using 1,3-dipolar azide-alkyne cycloaddition. The DNA template was produced by PCR, using an azidolabeled primer, and the self-priming moiety was attached to the immobilized DNA template by enzymatic ligation. Each cycle of SBS consists of the incorporation of the photocleavable fluorescent nucleotide into the DNA, detection of the fluorescent signal, and photocleavage of the fluorophore. The entire process was repeated to identify 12 continuous bases in the DNA template. These results demonstrate that photocleavable fluorescent nucleotide analogues can be incorporated accurately into a growing DNA strand during a polymerase reaction in solution and on a chip. Moreover, all four fluorophores can be detected and then efficiently cleaved using near-UV irradiation, thereby allowing continuous identification of the DNA template sequence. Optimization of the steps involved in this SBS approach will further increase the read-length. 2-nitrobenzyl linkerNA sequencing is a fundamental tool for biological science. 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 sequencing methods are needed to explore the complete human genome sequence for applications in clinical medicine and health care. Recent studies have indicated that an important route for identifying functional elements in the human genome involves sequencing the genomes of many species representing a wide sampling of the evolutionary tree (2). To overcome the limitations of the current electrophoresis-based sequencing technology (3-5), a variety of new DNA sequencing methods have been investigated. Such approaches include sequencing by hybridization (6), MS-based sequencing (7-9), and sequence-specific detection of single-stranded DNA by using engineered nanopores (10). More recently, DNA sequencing by synthesis (SBS) approaches such as pyrosequencing (11), sequencing of single DNA molecules (12), and polymerase colonies (13) have been widely explored.The concept of DNA SBS was revealed in 1988 (14). This approach involves detection of the identity of each nucleotide immediately after its incorporation into a growing strand of DNA in a polymerase reaction. Thus far, no complete success has been reported in using such a system to sequence DNA unambiguously. We proposed an SBS approach using photocleavable fluorescent nucleotide analogues on a surface in 2000 (15). In this approach, modified nucle...

We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.

DNA sequencing by synthesis (SBS) offers an approach for potential high-throughput sequencing applications. In this method, the ability of an incoming nucleotide to act as a reversible terminator for a DNA polymerase reaction is an important requirement to unambiguously determine the identity of the incorporated nucleotide before the next nucleotide is added. A free 3 -OH group on the terminal nucleotide of the primer is necessary for the DNA polymerase to incorporate an incoming nucleotide. Therefore, if the 3 -OH group of an incoming nucleotide is capped by a chemical moiety, it will cause the polymerase reaction to terminate after the nucleotide is incorporated into the DNA strand. If the capping group is subsequently removed to generate a free 3 -OH, the polymerase reaction will reinitialize. We report here the design and synthesis of a 3 -modified photocleavable fluorescent nucleotide, 3 -O-allyl-dUTP-PC-Bodipy-FL-510 (PC-Bodipy, photocleavable 4,4-difluoro-4-bora-3␣,4␣-diaza-s-indacene), as a reversible terminator for SBS. This nucleotide analogue contains an allyl moiety capping the 3 -OH group and a fluorophore Bodipy-FL-510 linked to the 5 position of the uracil through a photocleavable 2-nitrobenzyl linker. Here, we have shown that this nucleotide is a good substrate for a DNA polymerase. After the nucleotide was successfully incorporated into a growing DNA strand and the fluorophore was photocleaved, the allyl group was removed by using a Pd-catalyzed reaction to reinitiate the polymerase reaction, thereby establishing the feasibility of using such nucleotide analogues as reversible terminators for SBS. 2-nitrobenzyl linker ͉ photocleavageT he completion of the Human Genome Project (1, 2) has led to an increased demand for high-throughput and rapid DNA sequencing methods to identify genetic variants for applications in pharmacogenomics (3), disease gene discovery (4, 5), and gene function studies (6). Current state-of-the-art DNA sequencing technologies (7-11) to some extent address the accuracy and throughput requirements but suffer limitations with respect to cost and data quality. Thus, a new DNA sequencing approach is required to broaden the applications of genomic information in medical research and health care. In this regard, DNA sequencing by synthesis (SBS) offers an alternative approach to possibly address the limitations of current DNA sequencing techniques. We have previously described the design of a parallel chip-based SBS system, which uses a self-priming DNA template covalently linked to the glass surface of a chip and four modified nucleotides (12-14). The nucleotides are modified such that they have a photocleavable fluorescent moiety attached to the base (5 position of pyrimidines, 7 position of purines) and a chemically cleavable group to cap the 3Ј-OH. When the correct nucleotide is incorporated in a DNA polymerase reaction, specific to the template sequence, the reaction is temporarily terminated because of the lack of a free 3Ј-OH group. After the fluorescent signal is detected ...