Enzymatic shot-gun 5'-phosphorylation and 3'-sister phosphate exchange: a two-dimensional thin-layer chromatographic technique to measure DNA deoxynucleotide modification

J. J. Steinberg, Antonio Cajigas, Michael Brownlee

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

DNA adducts occur through environmental, therapeutic, dietary, oxygen stress, and aging processes. A modified thin-layer chromatographic (TLC) technique can assess base composition and adduct formation. This requires labeling DNA by "shot-gun" 5'-phosphorylation of representative 32P-α-deoxyribonucleotide monophosphates. Subsequent 3'-monophosphate digest "sister exchanges" a radioactive 32PO4 2- to the neighboring cold nucleotide. Separation in two-dimensional polyethyleneimine-cellulose TLC is carried out in acetic acid, (NH4)2SO4, and (NH4)HSO4. The technique was applied to control DNA, cold substition of dUMP, methylation, depurination, and pBR322. This technique quantifies low-molecular-mass adducts and DNA integrity both in vivo and in vitro.

Original languageEnglish (US)
Pages (from-to)41-55
Number of pages15
JournalJournal of Chromatography B: Biomedical Sciences and Applications
Volume574
Issue number1
DOIs
StatePublished - Feb 7 1992

ASJC Scopus subject areas

  • Chemistry(all)

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