2Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Division, Russian Academy of Sciences, pr. Akademika Lavrent'eva 9, 630090 Novosibirsk, Russia; fax: 330-4980
* To whom correspondence should be addressed.
Received October 27, 2004; Revision received December 14, 2004
A new, simple, and ingenious method for enzymatic synthesis of deoxy- and ribonucleoside-5´-triphosphates (dNTP and NTP, respectively) has been developed. The method includes the following stages: hydrolysis of DNA with DNase and immobilized S1-nuclease, phosphorylation of the resulting deoxy- and ribonucleoside-5´-monophosphates (dNMP and NMP, respectively) with nucleotidyl kinase from Escherichia coli, and purification by chromatography of the synthesized dNTP and NTP. dNMP was phosphorylated using an ATP-regenerating system based on acetokinase from E. coli and lithium acetylphosphate.
KEY WORDS: S1-nuclease, DNase, ribonucleoside-5´-triphosphate, deoxyribonucleoside-5´-triphosphate, immobilization, phosphorylation, DNA