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DNA Damage Quantification Kit-AP Site Counting-

Item # Unit Size
DK02-10
5 samples
DK02-12
20 samples

For Research Use Only Products

Application: Abasic site quantification in genomic DNA

- Determine the number of abasic sites in genomic DNA samples
- Colorimetric microplate assay
- Detection range: 1-40 abasic sites per 1x105 base pairs DNA

MSDS

Contents of the Kit: 5 samples

ARP Solution :100 μl x 1 tube     DNA Binding Solution  :10 ml x 1 bottle 
ARP-DNA standard solution :250 μl each Substrate Solution
:10 ml x 1 bottle
Filtration Tube :5 tubes    TE Buffer
:15 ml x 1 bottle
Washing Buffer :1 packet HRP-Streptavidin
:25 μl x 1 tube
96-well Microplate :1 plate    

Contents of the Kit: 20 samples

ARP Solution  :250 μl x 1 tube      DNA Binding Solution  :10 ml x 1 bottle 
ARP-DNA Standard Solution :250 μl each Substrate Solution :10 ml x 1 bottle
Filtration Tube :20 tubes TE Buffer :40 ml x 1 bottles
Washing Buffer :1 packet  HRP-Streptavidin :25 μl x 1 tubes
96-well Microplate :1 plate    

Storage Condition: 0-5ºC
Shipping Condition: ambient temperature or 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’-aminooxymethylcarbonylhydrazin-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.

Fig. 1 Mechanism of ARP Tagging at an Abasic Site


ARP Reaction (Preparation of ARP-labeled DNA)
1. Mix 10 μl of purified genomic DNA solution (100 μg per ml) and 10 μl ARP Solution in a 0.5 ml tube, and incubate at 37°C for 1 hour.
2. Wash the inside of the Filtration Tube with 100 μl of TE twice.
3. Add 380 μl TE to the reaction solution, and transfer the solution to a Filtration Tube.
4. Centrifuge the filtration tube at 2,500 g for 15 minutes, and discard the filtrate solution.
5. Add 400 μl TE to the filtration tube using a pipette to re-suspend the DNA on the filter with a pipette.
6. Centrifuge the Filtration Tube at 2,500 g for 15 minutes.a)
7. Add 200 μl TE to the Filtration Tube, using a pipette to re-suspend the DNA on the filter with a pipette.
8. Transfer the DNA solution to a 1.5 ml tube, and again add 200 μl of TE to the Filtration Tube to completely transfer the ARP-labeled DNA from the filter to the 1.5 ml tube.b)
9. Store the ARP-labeled genomic DNA solution at 0-5°C.
a) If the DNA solution still remains on the filter after centrifuging, centrifuge for another 5 minutes, and then proceed to step 7.
b) The recovery rate of DNA using a filtration tube is 90%, so the approximate concentration of ARP-labeled DNA is 2.25 μg per ml. For a more accurate determination of the number of abasic sites in sample DNA, we recommend measuring the actual DNA concentration.


Fig. 2 Isolation Process of ARP-labeled genomic DNA

Determination of the Number of Abasic Sites in DNA
Day 1:
1. Dilute 90 μl of the ARP-labeled genomic DNA with 310 μl TE.
2. Add 60 μl of ARP-DNA Standard Solution per well. Use 3 wells per 1 standard solution.
3. Add 60 μl of the diluted ARP-labeled genomic DNA solution per well. Use at least 3 wells per sample.
4. Add 100 μl DNA Binding Solution to each well and mix. Leave the plate at room temperature overnight.

Day 2:
5. Preparation of Solutions
Washing Buffer : Dissolve the contents of the Washing Buffer packet in 1 L of deionized or distilled water. Store this Washing Buffer at room temperature.
HRP-Streptavidin solution: Dilute HRP-Streptavidin with Washing Buffer to prepare 1/4000 diluted working solution.
6. Discard the DNA Binding Solution from all the wells and wash the wells five times with 250 μl of Washing Buffer.
    After discarding the Washing Buffer, invert the plate and tap it on a paper towel several times to completely remove the solution.
7. Add 150 μl of diluted HRP-Streptavidin working solution to each well and incubate the plate at 37°C for 1 hour.
8. Discard the solution in all wells, and wash the well five times with 250 μl of Washing Buffer.
9. Add 100 μl Substrate solution to each well and incubate at 37°C for 1 hour.
10. Measure the O.D. at 650 nm, and prepare a calibration curve using the data obtained from the ARP-DNA Standard Solution wells.
11. Determine the number of abasic sites in the genomic DNA using the calibration curve


Fig. 3 Typical calibration curve of DNA Damage Quantification Kit

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.




References
1. T. Lindahl, et al., Rate of Depurination of Native Deoxyribonucleic Acid. Biochemistry. 1972;11:3610-3618.
2. M. Liuzzi, et al., A New Approach to the Study of the Base-excision Repair Pathway Using Methoxyamine. J Biol Chem. 1985;260:5252-5258.
3. A. Sancar, et al., DNA Repair Enzymes. Annu Rev Biochem. 1988;57:29-67.
4. M. Weinfeld, et al., Response of Phage T4 Polynucleotide Kinase Toward Dinucleotides Containing Apurinic Sites: Design of a 32P-postlabeling Assay for Apurinic Sites in DNA. Biochemistry. 1990;29:1737-1743.
5. B. X. Chen, et al., Properties of a Monoclonal Antibody for the Detection of Abasic Sites, a Common DNA Lesion. Mutat Res. 1992;273:253-261.
6. J. A. Gralnick, et al., The YggX Protein of Salmonella enterica Is Involoved in Fe(II) Trafficking and Minimizes the DNA Damage Cause by Hydroxyl Radicals:Residue CYS-7 is Essential for YggX Function. J Biol Chem. 2003;278:20708-20715.
7. J. W. Pippin, et al., DNA Damage is a Novel Response to Sublytic Complement C5b-9 Induced Injury in Podocytes. J Clin Invest. 2003;111:877-885.
8. S. Watanabe, et al., Methylated DNA-binding Domain 1 and Methylpurine DNA Glycosylase Link Transcriptional Repression and DNA Repair in Chromatin. PNAS. 2003;100:12859-12864.
9. M. Endres, et al., Folate Deficiency Increases Postischemic Brain Injury. Stroke. 2005;36:321-325.
10. J. Li, et al., Angiotensin II-Induced Neural Differentiation via Angiotensin II Type 2 (AT2) Receptor-MMS2 Cascade Involving Interaction between AT2 Receptor-Interacting Protein and Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatase 1. Mol Endocrinol. 2007;21:499-511.
11. D. R. McNeill, et al., A Dominant-Negative Form of the Major Human Abasic Endonuclease Enhances Cellular Sensitivity to Laboratory and Clinical DNA-Damaging Agents. Mol Cancer Res. 2007;5:61-70.
12. C. A. Downs, et al., Cellular pathology and histopathology of hypo-salinity exposure on the coral Stylophora pistillata. Sci Total Environ. 2009;407:4838-4851.
13. C. A. Downs, et al., Symbiophagy as a cellular mechanism for coral bleaching. Autophagy. 2009;5:211-216.

Calibration Curve

Fig. 3 Typical calibration curve of DNA Damage Quantification Kit

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 ARPDNA Standard Solution is about 0.8-1.0. The O.D. value depends on HRP-Streptavidin activity.

FAQ
Can I use single-stranded DNA or RNA?
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.
How should genomic DNA be stored?
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.
How should I prepare the DNA?
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.
What should I do if the sample DNA concentration is less than 100 μg per ml?
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.
What should I do if the sample DNA is less than 1 μg?
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.
DK02