Enzymatic synthesis of oligodeoxyribonucleotides of defined sequence

Procedures for the controlled addition of one or more deoxyribonucleotide residues to the 3' end of an oligodeoxyribonucleotide primer are described. Polynucleotide phosphorylase (EC 2.7.7.8), purified from Escherichia coli B, catalyzes the reaction using a deoxyribonucleoside 5'-diphosphate as substrate, with Mn2+ as cofactor. Reaction occurs rapidly in aqueous solution, and no protecting groups are required, simplifying recovery and purification of the products. The concentrations of sodium chloride and manganous chloride in the incubation mixture are critical to obtaining good yield of the required product. Primers of chain length from 3 to 12 have been extended by up to 9 deoxyribonucleotide residues to obtain oligodeoxyribonucleotides of chain length up to 13. Yields of single addition products varied from 8 to 59%. Factors which influence these yields are discussed. The effects of added polyamines and some organic solvents on the reaction are described. Spermidine or dimethylsulfoxide in the incubation medium tend to favor the addition of several residues of deoxyribonucleotide to the primer.