Synthetic Polynucleotides

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylic acid residues to deoxyoligonucleotides. This reaction allows the specific labelling of deoxyoligonucleotides at the 3′‐ends if α‐ 32 PATP is used as substrate. In order to eliminate the product of the addition of two riboadenylic acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl 32 PpA r is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide‐3′‐ 32 Pmonophosphates corresponding to the 3′‐ends of the deoxyoligonucleotides. The riboadenosyl residues at the 3′‐termini of the monoaddition products also can be eliminated by treatment with periodate and cyclohexylamine whereupon deoxyoligonucleotides specifically labelled at the 3′‐termini with 32 P phosphomonoester groups are obtained. The sensitivity of the method and the faithfulness in the identification od the 3′‐terminal deoxynucleoside residues could be tested using as little as 0.1 A 260 units (5 μg) of oligodeoxynucleotides of specific base sequences.