A "cleanup procedure" involving periodate oxidation in the enzymatic synthesis of chemically pure alpha-32P and alpha-33P labelled deoxyribonucleotides.
Enzymatic synthesis of alpha-(32)P and alpha-(33)P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either gamma-(32)P adenosine triphosphate (gamma-ATP) or gamma-(32)P guanosine triphosphate (gamma-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 beta-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer gamma-(32)P ATP and alpha-(32)P 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 alpha-(32)P dATP synthesized by this process was assayed for its incorporation in lambda-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.
Muthukumaran
T
T
JONAKI, Labelled Biomolecules Laboratory, ILCJ, Board of Radiation and Isotope Technology, CCMB Campus, Uppal Road, Hyderabad- 500 007, Andhra Pradesh, India.
KrishnaMurthy
N V
NV
Sudhaharan
T
T
Muralidharan
B
B
D001345:Autoradiography; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D003854:Deoxyribonucleotides / Q000138:chemical synthesis*; D004798:Enzymes / Q000737:chemistry*; D010084:Oxidation-Reduction; D010504:Periodic Acid / Q000737:chemistry*
D003854:Deoxyribonucleotides; D004798:Enzymes; D010504:Periodic Acid