A sensitive biochemical assay for the detection of uracil.
We have developed a sensitive new assay for the detection of uracil in DNA. The assay described here is an adaptation to the highly sensitive aldehydic slot blot (ASB) assay developed by Nakamura et al. (Nakamura et al. 1998: Cancer Res 58:222-225) in which aldehydic DNA lesions (ADLs) are detected through binding of a biotinylated aldehydic reactive probe to DNA. The uracil DNA glycosylase (UDG)-coupled ASB assay uses uracil-DNA glycosylase to generate an abasic site, which is subsequently detected by the ASB methodology. The ability to modify this technique for the detection of uracil has these advantages: small quantities of DNA are required (4 microg of DNA); the assay is adaptable to DNA from both cells and tissues; sensitivity is as good as that achieved by less accessible methodologies, like gas chromatography-mass spectroscopy (GC-MS); DNA strand breaks are not a confounding variable; preexisting aldehydic lesions are blocked through the use of methoxyamine; variation is very low (<3%); radioactive isotopes are not required; and the assay is easy to establish and involves only equipment and reagents that are inexpensive and readily available. This assay is conceivably adaptable to the detection of other DNA base lesions through the use of a variety of DNA glycosylases.
Cabelof
Diane C
DC
Department of Nutrition and Food Science, Wayne State University, Detroit, Michigan, USA. d.cabelof@wayne.edu
Nakamura
Jun
J
Heydari
Ahmad R
AR
D000447:Aldehydes; D000818:Animals; D001681:Biological Assay; D003106:Colon / Q000737:chemistry; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D008099:Liver / Q000737:chemistry; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000737:chemistry; D013447:Sulfites / Q000494:pharmacology; D014498:Uracil / Q000032:analysis*; D051981:Uracil-DNA Glycosidase / Q000494:pharmacology
D000447:Aldehydes; D013447:Sulfites; D014498:Uracil; D004247:DNA; C427203:calf thymus DNA; D051981:Uracil-DNA Glycosidase; C042345:hydrogen sulfite