DNA sequencing by synthesis during a polymerase reaction using laser-induced fluorescence detection is an approach that has a great potential to increase the throughput and data quality of DNA sequencing. We report the design and synthesis of a photocleavable fluorescent nucleoside triphosphate, one of the essential molecules required for the sequencing-by-synthesis approach. We synthesized this nucleoside triphosphate by attaching a fluorophore, 4,4-difluoro-5,7-dimethyl-4-bora-3␣,4␣-diaza-s-indacene propionic acid (BODIPY), to the 5 position of 2 -deoxyuridine triphosphate via a photocleavable 2-nitrobenzyl linker. We demonstrate that the nucleotide analogue can be faithfully incorporated by a DNA polymerase Thermo Sequenase into the growing DNA strand in a DNA-sequencing reaction and that its incorporation does not hinder the addition of the subsequent nucleotide. These results indicate that the nucleotide analogue is an excellent substrate for Thermo Sequenase. We also systematically studied the photocleavage of the fluorescent dye from a DNA molecule that contained the nucleotide analogue. UV irradiation at 340 nm of the DNA molecule led to the efficient release of the fluorescent dye, ensuring that a previous fluorescence signal did not leave any residue that could interfere with the detection of the next nucleotide. Thus, our results indicate that it should be feasible to use four different fluorescent dyes with distinct fluorescence emissions as unique tags to label the four nucleotides (A, C, G, and T) through the photocleavable 2-nitrobenzyl linker. These fluorescent tags can be removed easily by photocleavage after the identification of each nucleotide in the DNA sequencing-by-synthesis approach.nucleoside triphosphate ͉ 2-nitrobenzyl linker ͉ photocleavage ͉ fluorescence ͉ DNA sequencing by synthesis T he ability to sequence DNA accurately and rapidly has revolutionized biology and medicine. The development of high-throughput genetic-analysis technologies involving chemistry, engineering, biology, and computer science has made it possible to move from studying single genes at a time to analyzing and comparing entire genomes. The current state-of-the-art technology for high-throughput DNA sequencing utilizes capillary array DNA sequencers with laser-induced fluorescence detection (1-4). Although capillary array DNA-sequencing technology addresses the throughput and read length requirements of large-scale DNA-sequencing projects to some extent, the sequencing throughput and accuracy required for disease-gene discovery and personalized medicine needs to be improved significantly. For example, electrophoresis-based DNAsequencing methods have difficulty detecting heterozygotes unambiguously and are not 100% accurate in regions rich in nucleotides comprising guanine or cytosine because of compression (5, 6). In addition, the first few bases after the priming site are often masked by the high fluorescence signal from excess dye-labeled primers or dye-labeled terminators and are difficult to identify. Thus, ...