A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion.
Free radicals produce a wide spectrum of damages; among these are DNA base damages and abasic (AP) sites. Although several methods have been used to detect and quantify AP sites, they either are relatively laborious or require the use of radioactivity. A novel reagent for detecting abasic sites in DNA was prepared by reacting O-(carboxymethyl)hydroxylamine with biotin hydrazide in the presence of carbodiimide. This reagent, called Aldehyde Reactive Probe (ARP), specifically tagged AP sites in DNA with biotin residues. The number of biotin-tagged AP sites was then determined colorimetrically by an ELISA-like assay using avidin/biotin complex conjugated to horseradish peroxidase as the indicator enzyme. With heat/acid-depurinated calf thymus or bacteriophage f1 DNA, ARP detected femtomoles of AP sites in DNA. Using this assay, DNA damages generated in calf thymus, phi X174 RF, and f1 single-stranded DNA, X-irradiated in phosphate buffer, were easily detectable at 10 rad (0.1 Gy). Furthermore, ARP sites were detectable in DNA isolated from heat-inactivated X-irradiated (10 Gy) and methyl methanesulfonate (MMS)-treated (5 microM) Escherichia coli cells. The rate of production of ARP sites was proportional to the X-ray dose as well as to the concentration of MMS. Thus, the sensitivity and simplicity of the ARP assay should provide a potentially powerful method for the quantitation of AP sites or other DNA lesions containing an aldehyde group.
Kubo
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Department of Microbiology and Molecular Genetics, Markey Center for Molecular Genetics, University of Vermont, Burlington, Vermont 05405.
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D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives* / Q000737:chemistry; D002417:Cattle; D004247:DNA / Q000737:chemistry / Q000528:radiation effects; D004249:DNA Damage; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005609:Free Radicals; D007202:Indicators and Reagents; D008741:Methyl Methanesulfonate / Q000633:toxicity; D009692:Nucleic Acid Heteroduplexes
D005609:Free Radicals; D007202:Indicators and Reagents; D009692:Nucleic Acid Heteroduplexes; C074188:O-(biotinylcarbazoylmethyl)hydroxylamine; C053311:biotin hydrazide; D001710:Biotin; D004247:DNA; D008741:Methyl Methanesulfonate