Synthetic Polynuccleotides.

The enzyme terminal deoxynucleotidyl transferase catalyzes the addition of one or two riboadenylie acid residues to deoxyoligonucleotides. This reaction allows the specific labeling of deoxyoligonucleotides at the 3′-ends if [α-³²P]ATP is used as substrate. In order to eliminate the product of the addition of two riboadenylie acid residues the total reaction mixtures after incubation with terminal transferase are treated with alkali and subsequently with phosphatase. Deoxyoligonucleotidyl [³²P]pAr is then obtained as the sole reaction product. Digestion with spleen phosphodiesterase yields the deoxynucleotide-3′-[³²P]monophosphates 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 labeled at the 3′-termini with [³²P]phosphomonoester groups are obtained. The sensitivity of the method and the faithfulness in the identification of the 3′-terminal deoxynucleoside residues could be tested using as little as 0.1 A₂₅₀ units (5 μg) of oligodeoxynucleotides of specific base sequences. [ABSTRACT FROM AUTHOR]