Enzymatic synthesis of deoxyATP using DNA as starting material

The authors describe the synthesis of deoxyadenosine triphosphate (dATP) from DNA on a 200-mmol scale. Enzymatic digestion of DNA to a mixture of deoxynucleoside monophosphates was accomplished by a two-step process: initial conversion to a mixture of oligonucleotides using soluble DNase I, and subsequent hydrolysis of this mixture to mononucleoside monophosphates using nuclease P sub(1) immobilized in polyacrylamide gel. The overall conversion of the deoxynucleotide moieties in the original DNA to soluble deoxynucleoside monophosphates was 75-90%. Selective conversion of dAMP to dATP in the presence of a mixture of dAMP, dGMP, dCMP, and TMP was accomplished by enzymatic phosphorylation using PEP, a catalytic quantity of ATP, adenylate kinase, and pyruvate kinase. DeoxyATP was isolated from the reaction mixture as its barium salt in 67% yield and 60% purity. A subsequent simple purification yielded dATP with 95% purity, in 40% overall yield based on dAMP moieties present in the starting DNA.