TY - JOUR
T1 - Enzymatic shot-gun 5'-phosphorylation and 3'-sister phosphate exchange
T2 - a two-dimensional thin-layer chromatographic technique to measure DNA deoxynucleotide modification
AU - Steinberg, J. J.
AU - Cajigas, Antonio
AU - Brownlee, Michael
N1 - Funding Information:
Our thanks are due to Drs . Julius Marmur, Sam Seifter, and Vern Schramm for their counsel and critiques, to Dr . Sidney Goldfischer for support, to Dr . William Franklin for review, to Dr . Michael Brenowitz for discussions on densitometry, and to Dr . Tom Leyh for discussions regarding Ambis scintillation counting . The understanding and help of Sari Miller, Simone, Rachel, and Abigail Steinberg is appreciated . In honor of the retirement of Drs . Arthur and Betsy Upton, the anniversary of Moses and Clara Steinberg, and the birthday of Dr . Julius Marmur . This work was supported in part by the American Diabetes Association and the American Federation for Aging Research . A .C. is a Fellow of the New York State Health Research Council .
PY - 1992/2/7
Y1 - 1992/2/7
N2 - 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.
AB - 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.
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U2 - 10.1016/0378-4347(92)80096-9
DO - 10.1016/0378-4347(92)80096-9
M3 - Article
C2 - 1629287
AN - SCOPUS:0026548505
VL - 574
SP - 41
EP - 55
JO - Journal of Chromatography B: Biomedical Sciences and Applications
JF - Journal of Chromatography B: Biomedical Sciences and Applications
SN - 0378-4347
IS - 1
ER -