Substrate Properties of Fluorescent Ribonucleotides in the Terminal Transferase-Catalyzed Labeling of DNA Sequencing Primers

Abstract

Terminal deoxynucleotidyltransferase (terminal transferase, E.C. 2.7.7.31) has been used to add a single fluorescent ribonucleotide to the 3′ terminus of DNA sequencing primers, thereby creating primers suitable for automated DNA sequence analysis. The previously introduced procedure using fluorescein-UTP for the postsynthetic labeling of primers can, under appropriate reaction conditions, now be extended to commercially available fluorescein- ATP and fluorescein-CTP permitting greater flexibility in primer design. The products of these addition reactions have been shown to provide sequence data qualitatively and quantitatively identical to those obtained with conventional 5′-terminally labeled primers using cycle sequencing conditions in conjunction with an automated sequencer. Ribonucleotide derivatives of four other dyes (coumarin, tetramethylrhodamine, lissamine and Texas Red®) were also examined for their potential in the terminal transferase-catalyzed reaction. Whereas coumarin-UTP was efficiently incorporated giving a monoaddition product, the conjugates of all other dyes with ATP, CTP and UTP were extremely poor substrates under all conditions tested.