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IMPROVED THIN-LAYER CHROMATOGRAPHIC SEPARATION OF 32P-POSTLABELING DNA ADDUCTS
Citation:
Spencer, G., A. Beach, AND R. Gupta. IMPROVED THIN-LAYER CHROMATOGRAPHIC SEPARATION OF 32P-POSTLABELING DNA ADDUCTS. U.S. Environmental Protection Agency, Washington, D.C., EPA/600/J-93/343 (NTIS PB93229615), 1993.
Description:
DNA adducts represent the putative initiating event in the chemical process. 2P-Postlabeling is one of several assayswhich have been developed for the sensitive detection of DNA adducts. n integral part of the 32p-postlabeling assay is the separation of adducted nucleotides by multidirectional, multisolvent, anion-exchange polyethleneimine-cellulose thin-layer chromatography. tandard since the introduction of this assay it has been the use of high-Wt, high-una solvents for the resolution of adducts during the D3 and D4 phases of the chromatography. rea solvents are able to separate adducts resulting from a number of chemicals, however, they are time-consuming, retain a lot of background noise, may push adducts into inadequately resolved diagonal radioactive zones, and may not separate adducts of similar structure. n this study we introduce the use of a dilute ammonium hydroxide solvent for D4 chromatography and compare it to other standard solvents such as lithium chloride-Tris. CL-urea sodium phosphate-Tris HCL-urea, and isopropanol-4 M ammonium hydroxide for adduct separation, resolution, recovery, retention of background noise, and chromatography development time. e found that 0.2 M ammonium hydroxide worked well for the recovery, separation, and resolution of a wide array of adducts derived from highly lipophilic polycyclic aromatic hydrocarbons and aromatic amines. n addition, this solvent required much less time (<1/4) as compared to the other solvents and more importantly allowed the separation of adducts which otherwise comigrated and were not visible when using the other three D4 solvents. ackground radioactivity per cm2 of the thin layers was also reduced two- to three-fold with the ammonia solvent versus the urea-based solvent. hus, the use of this dilute ammonia system provides powerful resolution in a time-efficient manner.
