Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry

Abstract

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...

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