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A sensitive assay for determining the specific activity of highly labeled 2′-deoxyribonucleoside 5′-[α-32P]triphosphates

✍ Scribed by Harry R. Burrell; Laszlo Beres

Publisher
Elsevier Science
Year
1980
Tongue
English
Weight
645 KB
Volume
106
Category
Article
ISSN
0003-2697

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✦ Synopsis

A procedure is described for obtaining the specific activity of c@P-labeled nucleotides in the range where mass determination by uv spectrophotometry or by radioisotopic double-label techniques is not feasible. The procedure, based on the template-dependent synthesis of an alternating-sequence copolymer catalyzed by DNA polymerase, requires no elaborate equipment and uses commercially available materials and enzymes. Results obtained with this assay indicate that the measured specific activities of 2'-deoxyribonucleoside 5'-[@P]triphosphates synthesized according to published procedures can vary significantly from expected theoretical values. I Abbreviations used: poly(dA-dT), altemating-sequence copolymer of deoxyadenosine monophosphate and deoxythymidine monophosphate; poly(dG-dC), alternating-sequence copolymer of deoxyguanosine monophosphate and deoxycytidine monophosphate; poly(dI-dC), alternating-sequence copolymer of deoxyinosine monophosphate and deoxycytidine monophosphate; DTT, dithiothreitol; dNTP, 2'-deoxyribonucleoside S-triphosphate; dNMP, 2'-deoxyribonucleoside 5'-monophosphate; PEI, polyethyleneimine; tic, thin-layer chromatography.

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