; 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...