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

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.