Labeling the 3′ Termini of Oligonucleotides Using Terminal Deoxynucleotidyl Transferase

Abstract

Terminal deoxynucleotidyl transferase (TdT, also simply called terminal transferase) is a template-independent polymerase that catalyzes the addition of deoxynucleotides and dideoxynucleotides to the 3′-hydroxyl terminus of a DNA molecule. Cobalt (Co²⁺) is a necessary cofactor for the activity of this enzyme. Incorporation at the 3′ terminus can be limited to just 1 nt by using [α-³²P]ddATP or biotin-, digoxigenin (DIG)-, or fluorescein-ddUTP. Because none of these molecules carries a 3′-hydroxyl group, no additional molecules can be incorporated. Alternatively, the enzyme is capable of adding several (2–100) nt to 3′ ends in a so-called homopolymeric “tailing” reaction. A tailing reaction is performed in the presence of a mixture of labeled and unlabeled dNTPs. The rate of addition of dNTPs, and thus the length of the tail, is a function of the ratio of 3′ DNA ends to dNTP concentration and, in addition, the specific dNTP that is used.