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.