Enzymatic synthesis of oligonucleotides of defined sequence. Addition of short blocks of nucleotide residues to oligonucleotide primers

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Abstract
Polynucleotide phosphorylase from Escherichia coli can be used to catalyse the addition of short tracts of deoxyadenylate residues to the 3′-termini of deoxyribooligonucleotides of the type pdAn-dN (where dN = dC, dT or dG) using dADP as donor. Similarly, the enzyme can also be used to catalyse the addition of short tracts of adenylate residues to the 3′-termini of ribooligonucleotides of the type An-N (where N = C, U or G) using ADP as donor. In the riboo-ligonucleotide series, phosphorolytic cleavage of the primer oligonucleotides is significant and results in the concommitant production of oligoadenylates lacking the N residue. Oligomers of the same length, with and without the residue N, were readily separated by thermal elution from cellulose-pdT9 columns. This latter procedure therefore provides a simple method for purification of the oligoadenylates containing an internal base substitution and it also provides a convenient assay for oligonucleotide phosphorolysis.