A procedure for the measurement of intracellular deoxyribonucleoside triphosphate pools by thin layer chromatography

✍ Scribed by Charles D. Yegian

• Publisher: Elsevier Science
• Year: 1974
• Tongue: English
• Weight: 449 KB
• Volume: 58
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(74)90462-x

✦ Synopsis

Rapid quantitative measurement of deoxyribonucleoside triphosphate (dNTP) pools has been difficult. Systems measuring the incorporation of radioactive ribose and base precursors are limited by the rate and extent of equilibration with cellular pools. The use of 32P has been difficult as a result of the incorporation of orthophosphate into many compounds other than nucleotides. Thin layer chromatography on PEI-cellulose' has been found to be a sensitive technique for separating nucleotides (1). However, the systems described have several disadvantages including "tailing" of 32P on the chromatogram and interference from the other 32Plabeled compounds. Sensitive enzymatic assays have been developed recently for pool size measurements of deoxyribonucleoside triphosphates (2-4). Described below is a quick, easy, and more sensitive technique for the determinat.ion of dNTP pool leveis in growing cells.

MATERIALS AND GENERAL METHODS

Materials. Nonradioactive nucleotides were obtained from Sigma Chemical Co., St. Louis, MO. Carrier-free orthophosphoric acid, H,3?P0,, in 0.02 N HCl, was obtained from ICN, Irvine, CA. The purity of each lot of H,32P0, was determined as described below. Poly(ethylene)imine was obtained from Chemirad Corp., East Brunswick, NJ. Cellulose powder MN 300, made by Macherey, Nagel, and Co., was purchased from Brinkmann Instruments, Westbury, NY. Sheets of Bakelite Rigid Vinyl (type VSA 3310, Clear 31, Matte 06, 0.010 in. thick) were shipped flat from Commercial Plastics and Supply Corp., New York. Autoradiography ' Deceased. ' Abbreviation used: PEI-cellulose, poly(ethylene)imine-cellulose; tic, thin layer chromatography; dNMP, dNDP, dNTP, deoxyribonucleoside mono-, di-, and triphosphates, respectively; Tris, tris(hydroxymethyl)aminomethanc.