Oxidative damage to DNA is a result of its interaction with reactive oxygen species (ROS), in particular, the hydroxy radical. Hydroxy radicals, which are produced from superoxide anion and hydrogen peroxide by the Fenton reaction, produce multiple modifications in DNA. Oxidative attacks by hydroxy radicals on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple abasic sites (AP sites). In fact, AP sites are one of the major types of damage generated by ROS. Aldehyde Reactive Probe (ARP; N Eaminooxymethylcarbonylhydrazin-D-biotin) reacts specifically with an aldehyde group present on the open ring form of the AP sites (Fig. 1). This reaction makes it possible to detect DNA modifications that result in the formation of an aldehyde group. After treatment with excess ARP reagent, all of the AP sites on DNA are tagged with a biotin residue. These biotin-tagged AP sites can be quantified using the avidin-biotin assay, followed by colorimetric detection with either peroxidase or alkaline phosphatase conjugated to the avidin. DNA Damage Quantification Kit contains all the necessary solutions for detecting between 1 to 40 AP sites per 1 x 105 base pairs.
AP site Detection Principle
Mechanism of ARP Tagging at an Abasic Site
How to Prepare a Calibration Curve
1. Calculate the average O.D. of each ARP-DNA standard solution.
2. Subtract the blank O.D. from the average O.D.a)
3. Plot the O.D. corresponding to the number of AP sites of the standard solution. X-axis is the number of AP sites and Y-axis is the O.D.
4. Determine the number of AP sites in the sample using this calibration curve.
a) The blank O.D. is about 0.04-0.06 and the O.D. of the 40 ARP DNA standard solution is about 0.8-1.0. The O.D. value depends on HRPStreptavidin activity.
Fig. 3 Typical calibration curve of DNA Damage Quantification Kit
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