Enzymic Synthesis of Polynucleotides

The formation of mono- and diaddition products resulting from incorporation of rAMP residue(s) at the 3' end of an oligodeoxynucleotide primer catalyzed by the enzyme terminal deoxynucleotidyltransferase is strongly influenced by the primer to substrate ratio in the reaction mixture. Low concentrations (1 to 100 µm) of primer (hexathymidylate) and an excess (1000 µm) of substrate (rATP) cause an exclusive synthesis of the diaddition product. The monoaddition product, undetectable at a very low primer to substrate ratio, however, becomes detectable when the primer concentration approaches one-fifth of the concentration of substrate. In the presence of an excess of primer, mainly the monoaddition product is formed. The utilization of primer can be driven to completion using an excess (1 mm) of rATP and a limited (50 to 100 µm) concentration of primer. The optimum substrate utilization is noticed when there is a 4- to 5-fold excess of primer in the incubation medium. All four common ribonucleotides (rAMP, rGMP, rCMP, rUMP) can be added at the 3' end of an oligodeoxynucleotide primer. Such ribonucleotide terminated primers can then be employed for the initiation of new chains at the ribo-3' terminus of the primer, using all four common deoxynucleoside triphosphates (dATP, dGTP, dCTP, dTTP) giving rise to polydeoxynucleotides covalently linked to the original deoxyoligonucleotide primer by a specific ribonucleotide linkage.