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DNA Damage Quantification Kit-AP Site Counting-

Item # Unit Size
20 samples

For Research Use Only Products

Abasic Site Quantification in Genomic DNA
∼ Feature ∼
- Determine the number of abasic sites in genomic DNA samples
- Colorimetric microplate assay
- Detection range: 1-40 abasic sites per 1x105 base pairs DNA

Contents of the Kit: 20 samples

Storage Condition: 0-5ºC
Shipping Condition: with blue ice

Required Equipment and Materials
microplate reader with 650 nm filter, incubator, microcentrifuge, 10 μl and 200 μl adjustable pipettes, multi-channel pipette

Product Description
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

Fig. 1 Mechanism of ARP Tagging at an Abasic Site
Recent Publications
Samples from Treatments References
MEF cells Cisplatin, Oxaliplatin Novel Role of Base Excision Repair in Mediating Cisplatin Cytotoxicity A. Kothandapani, et al., J Biol Chem, 286, 14564(2011)
SF767 glioblastoma AP endonuclease repair inhibitor Novel Small-Molecule Inhibitor of Apurinic/Apyrimidinic Endonuclease 1 Blocks Proliferation and Reduces Viability of Glioblastoma Cells A. Bapat, et al., J Pharmacol Exp Ther, 334, 988(2010)
hippocampal tissue global cerebral ischemia Apurinic/apyrimidinic endonuclease APE1 is required for PACAP-induced neuroprotection against global cerebral ischemia R. A. Stetler, et al., PNAS, 107, 3204(2010)
CHO cells dominant-negative form of AP endonuclease 1 expressing Impairment of APE1 Function Enhances Cellular Sensitivity to Clinically Relevant Alkylators and Antimetabolites D. R. McNeill, et al., Mol Cancer Res, 7, 897(2009)
C57BL/6J mouse Valsartan Temporary Pretreatment With the Angiotensin II Type 1 Receptor Blocker, Valsartan, Prevents Ischemic Brain Damage Through an Increase in Capillary Density J. Li, et al., Stroke, 39, 2029(2008)

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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9) M. Endres, et al., Folate Deficiency Increases Postischemic Brain Injury. Stroke. 2005;36:321-325.
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Q. Can I use single-stranded DNA or RNA?
A. No, you cannot use this kit to determine the number of abasic sites in single-stranded DNA or RNA. The O.D. reading of single-stranded DNA will be nearly twice that of double-stranded DNA because of the binding efficiency on the microplate.

Q. How should genomic DNA be stored?
A. Prepare a DNA pellet and store at -20°C or -80°C if the DNA cannot be labeled with ARP immediately after isolation. After ARP labeling, the sample can be stored at 4°C in TE Buffer for several months.

Q. How should I prepare the DNA?
A. You can use general protocols or commercially available DNA isolation kits. Between 2 to 4 abasic sites per 1 x 105 base pairs will be created during the DNA isolation process. Therefore, use the same isolation method to prepare each DNA sample.

Q. What should I do if the sample DNA concentration is less than 100 μg per ml?
A. You can either use a filtration tube to concentrate your sample DNA or ethanol precipitation to recover DNA as a pellet and then re-dissolve it to prepare a 100 μg per ml solution.

Q. What should I do if the sample DNA is less than 1 μg?
A. Add the same volume of ARP Solution and follow the manual. The recovery of the ARP-labeled DNA may be lower than the usual reactions, so measure the ARP-labeled DNA solution. The average recovery rate of the 0.5 μg DNA and 0.25 μg DNA is 70% and 50%, respectively.