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A “Cleanup Procedure” involving periodate oxidation in the enzymatic synthesis of chemically pure α-32P and α-33P labelled deoxyribonucleotides by T. Muthukumaran; N.V. KrishnaMurthy; T. Sudhaharan; B. Muralidharan is a Physics and Astronomy article available to read on EtoBox.

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Enzymatic synthesis of a-32 P and a-33 P labelled deoxyribonucleotides involves the transfer of radiolabelled phosphorus from either g-32 P adenosine triphosphate (g-ATP) or g-32 P guanosine triphosphate (g-GTP). Subsequent removal of these ribonucleotides is essential for the preparation of chemically pure deoxyribonucleotides. Agarosephenyl 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 b-elimination using alkali. The product was purified using anion exchange chromatography. The efficiency of the process was validated using tracer g-32 P ATP and a-32 P dATP. Samples at various steps were analysed by TLC, autoradiography and HPLC. During the process ATP is oxidised whereas 2 0 -deoxyadenosine tripho

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Author

T. Muthukumaran; N.V. KrishnaMurthy; T. Sudhaharan; B. Muralidharan

Publisher

Elsevier Science; Elsevier ; Elsevier Ltd.; Elsevier BV (ISSN 0969-8043)

Published

2005

Language

EN

Field

Physics and Astronomy (Physical Sciences)