Analysis of uracil in DNA by gas chromatography-mass spectrometry.
A sensitive method using gas chromatography and negative chemical ionization mass spectrometry for the detection of uracil in DNA is described. Uracil DNA glycosylase is used to specifically cleave uracil from DNA. Once removed, uracil is derivatized with 3,5-bis(trifluoromethyl)benzyl bromide and the  sample components are separated with gas chromatography. Negative chemical ionization mass spectrometry is used to quantitatively detect as little as 1 pg of uracil per 100 micrograms DNA. This assay is 10 times more sensitive than previously described quantitative methods for the detection of uracil in DNA. We apply this method as part of a project to determine why folic acid deficiency causes DNA breaks. We demonstrate that inhibition of folic acid metabolism induces an accumulation of uracil in DNA.
Blount
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BC
Department of Molecular and Cell Biology, University of California, Berkeley 94720.
Ames
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BN
D000818:Animals; D001593:Benzyl Compounds; D004247:DNA / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004269:DNA, Bacterial / Q000737:chemistry; D004926:Escherichia coli; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D006498:Hepatectomy; D007202:Indicators and Reagents; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008115:Liver Regeneration; D008297:Male; D008727:Methotrexate / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D014498:Uracil
D001593:Benzyl Compounds; D004269:DNA, Bacterial; D007202:Indicators and Reagents; C089218:3,5-bis(trifluoromethyl)benzyl bromide; D014498:Uracil; D004247:DNA; D008727:Methotrexate