A “Cleanup Procedure” involving periodate oxidation in the enzymatic synthesis of chemically pure α-32P and α-33P labelled deoxyribonucleotides

Enzymatic synthesis of α-32P and α-33P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either γ-32P adenosine triphosphate (γ-ATP) or γ-32P guanosine triphosphate (γ-GTP). Subsequent removal of these ribonucleotides is essential for the preparation of chemically pure deoxyribonucleotides. Agarose-phenyl boronate columns, which bind specifically to cis-diol moieties, have been used for the removal of ribonucleotide contaminants. However, this involves column losses and additional radiation exposure. In the present work we describe a chemical method for the improvement of the chemical purity, based on the preferential oxidation of ribose sugars by periodate. The cis-diol moiety of ribose is specifically oxidised to the dialdehyde. The excess periodate ions were destroyed using ethylene glycol. The phosphate group was then cleaved by β-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer γ-32P ATP and α-32P dATP. Samples at various steps were analysed by TLC, autoradiography and HPLC. During the process ATP is oxidised whereas 2′-deoxyadenosine triphosphate (dATP) remains intact. The α-32P dATP synthesized by this process was assayed for its incorporation in λ-DNA by the random priming method and was found to be effectively incorporated. The process developed is an efficient and convenient method for the preparation of chemically pure deoxyribonucleotides.