METHOD FOR THE ENZYMATIC-SYNTHESIS AND PURIFICATION OF [ALPHA-P-32] NUCLEOSIDE TRIPHOSPHATES

A simplified method is described for the enzymatic synthesis and purification of [α-32P]ribo- and deoxyribonucleoside triphosphates. The products are obtained at >97% radiochemical purity with yields of 50-70% (relative to 32Pi) by a two-step elution from DEAE-Sephadex. All reactions are done in one vessel as there is no need for intermediate product purifications. This method is therefore suitable for the synthesis of these radioactive compounds on a relatively large scale.The sequential steps of the method involve first the synthesis of [γ-32P]ATP and the subsequent phosphorylation of nucleoside 3′ monophosphate with T4 polynucleotide kinase to yield nucleoside 3′,[5′-32P]diphosphate.Hexokinase is used after the T4, reaction to remove any remaining [γ-32P]ATP. Nucleoside 3′,[5′-32P]diphosphate is treated with nuclease P-l to produce the nucleoside [5′-32P]monophosphate which is phosphorylated to the [α-32P] nucleoside triphosphate with pyruvate kinase and nucleoside monophosphate kinase. Adenosine triphosphate used as the phosphate donor for [α-32P] deoxynucleoside triphosphate syntheses is readily removed in a second purification step involving affinity chromatography on boronate-polyacrylamide. [α-32P]Ribonucleoside triphosphates can be similarly purified when deoxyadenosine triphosphate is used as the phosphate donor. © 1979 Information Retrieval Limited.