3'-O-Azidomethyl-dUTP is an excellent dNTP building block that can be used for developing DNA detecting probes for DNA sequencing by synthesis (SBS) and other molecular biology applications. DNA sequencing is a fundamental tool in biological and medical research. A variety of DNA sequencing methods have been developed in recent years. Currently, the dominant method is sequencing by synthesis (SBS), an approach that determines DNA sequences during the polymerase reaction. The most widely used high-throughput SBS technology uses cleavable fluorescent reversible nucleotide terminator sequencing chemistry. The reversible terminator sequencing chemistry is based on the principle: each of the four nucleotides (A, C, G, T) is modified by attaching a unique cleavable fluorophore to the base and capping the 3’-OH group with a small reversible chemical moiety so that they are still recognized by DNA polymerase as substrates. The fluorescence based SBS suffers limited read length since the current cleavable fluorescent dNTPs used in SBS leave a large fluorescence tag on the base of the growing DNA strand. Conceptually, the much smaller azidomethyl group on the dNTPs may be used as a tag for sequencing. Due to the small size of the azido label, the 3’-O-azidomethyl-dNTPs are efficient substrates for DNA polymerase. In the SBS cycles, the natural nucleotides are restored after each incorporation and cleavage, producing a growing DNA strand that bears no modifications and will not impede further polymerase reactions.