Highly fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate as an efficient substrate for DNA polymerases.

We herein describe the synthesis of fluorescent 5-(5,6-dimethoxybenzothiazol-2-yl)-2'-deoxyuridine 5'-triphosphate (d(bt)UTP) and primer extension reactions using d(bt)UTP. We also carried out primer extension reactions using the (bt)U template. B family DNA polymerases, such as KOD, Deep Vent (exo-), and 9°N(m) DNA polymerases, were effective for elongation with d(bt)UTP. Deep Vent (exo-) and KOD DNA polymerases have excellent fidelity for incorporating d(bt)UTP only at the site opposite the adenine template and only dATP when using the (bt)U template. Therefore, d(bt)UTP is an excellent fluorescent nucleotide that can be incorporated into DNA by DNA polymerases.