Incorporation of reporter molecule‐labeled nucleotides by DNA polymerases. I. Chemical synthesis of various reporter group‐labeled 2′‐deoxyribonucleoside‐5′‐triphosphates

Fluorescent-labeled DNA is generated through enzymatic incorporation of fluorophore-linked 2′-deoxyribonucleoside-5′-triphosphates (dNTPs) by DNA polymerases. We describe the synthesis of a variety of dye-labeled dNTPs. Amino-linker-modified 5′-triphosphates of all four naturally occurring nucleobases were used as precursors. Commercially available dyes were coupled to the amino function of the side chain. In addition, we attached novel fluorophore derivatives. The labeled products were obtained in at least 96% purity after HPLC purification. Enzymatic incorporation into DNA and subsequent extension of the modified DNA chain were studied. VentR exo– DNA polymerase and a defined template–primer system were used to analyze each dye-labeled dNTP derivative. Our data suggest that the incorporation efficiency depends on the selected dye, the nucleobase or a combination of both.