An improved procedure for utilizing terminal transferase to add homopolymers to the 3′ termini of DNA

Abstract

Terminal deoxynucleotidyl transferase (E.C.2.7.7.3.1.) from calf thymus was used to add homopolymer tails to duplex DNA with 3′ protruding, even, or 3′ recessive ends. A gel electrophoresis method was employed to analyze the tail length and the percent of DNA with tails. In all the tailing reactions, dA, dT, and dC tails from CoCl₂-containing buffer were longer than those from MnCl₂– or MgCl₂–-containing buffers, whereas dG tails from MnCl₂–containing buffer were the longest. By varying the ratio of dNTP over DNA terminus and the concentration of terminal transferase, optimal conditions were found for adding dG or dC tails of 10–25 nucleotides in length and dA and dT tails of 20–40 nucleotides in length to duplex DNA with all types of 3′ termini.