Synthetic Polynucleotides
The Terminal Addition of Riboadenylic Acid to Deoxyoligonucleotides by Terminal Deoxynucleotidyl Transferase as a Tool for the Specific Labelling of Deoxyoligonucleotides at the 3′‐Ends
Definition. A260 unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in a 1‐cm path length cell.
Abstract
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‐32P]ATP 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 [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 labelled at the 3′‐termini with [32P] 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 A260 units (5 μg) of oligodeoxynucleotides of specific base sequences.
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