DK02-10

DNA Damage Quantification Kit -AP Site Counting- Unit: 5 samples

Description:

** ARP-DNA Standard Solution contains the followings:
         0 ARP-DNA Standard Solution 250 ul x 1
      2.5 ARP-DNA Standard Solution 250 ul x 1
         5 ARP-DNA Standard Solution 250 ul x 1
       10 ARP-DNA Standard Solution 250 ul x 1
       20 ARP-DNA Standard Solution 250 ul x 1
       40 ARP-DNA Standard Solution 250 ul x 1

Oxidative damage to DNA is a result of the interaction of DNA with reactive oxygen species (ROS), in particular, the hydroxy radical which is converted from superoxide and hydrogen peroxide by the Fenton reaction. Hydroxy radicals produce a multiplicity of modifications in DNA. Oxidative attack by hydroxy radical on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple AP sites. In fact, they are one of the major types of damage generated by ROS. It has been estimated that endogeneous ROS can result in about 2x10⁵ base lesions per cell per day.

Aldehyde Reactive Probe (ARP) reagent (N'-aminooxymethylcarbonylhydrazino-D-biotin) reacts specifically with an aldehyde group which is the open ring form of the AP sites. This reaction makes it possible to detect DNA modifications that result in the formation of an aldehyde group. After treating DNA containing AP sites with ARP reagent, AP sites are tagged with a biotin residue. By using an excess amount of ARP, all AP sites can be converted to biotin-tagged AP sites. Therefore, AP sites can be quantified using avidin-biotin assay followed by a colorimetric detection of peroxidase or alkaline phosphatase conjugated to the avidin.

DNA Damage Quantification Kit is for quantification of abasic sites in genomic DNA, which corresponds to colorimetric 96-well microplate assay. ARP standard DNA solutions in this kit are prepared by heat/acid depurination (Lindahl & Nyberg, 1972, Kubo, et al, 1992) of calf thymus DNA to control the number of abasic sites from 0 to 40 per 1x10^5 bp. These DNA solutions are treated with ARP and purified. Standard 0 APP-DNA is prepared by methoxyamine-treated calf thymus DNA without heat/acid depurination. Since this kit contains centrifugal filtration tubes for the purification of ARP-labeled sample DNA, there is no need to determine the DNA concentration after the ARP reaction.

Standard ARP DNA and purified ARP-treated sample DNA are fixed on the 96-well plate with DNA Binding Solution. The number of the abasic sites in the sample DNA can then be determined by the biotin-avidin-peroxidase assay. Since the standard ARP DNAs are double stranded, this kit is not applicable to the abasic sites detection of single stranded DNA. The O.D. value of a single stranded DNA sample is nearly twice as high as that of a double stranded DNA sample under the same assay condition. For the isolation of genomic DNA from samples, a guanidine/detergent-based DNA isolation method is recommended.

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/ml) and 10 µl ARP solution in a 0.5 ml tube, and incubate at 37 ºC for 1 hour.
2. Wash the inside of Filtration tube cup with 100 µl TE.
3. Add 380 µl TE to the ARP-labeled DNA solution, and transfer the solution to Filtration tube.^a)
4. Centrifuge Filtration tube at 5,000 g for 15 min, and discard the filtrate solution.
5. Add 400 µl TE to Filtration tube, and re-suspend the DNA on the filter with pipetting.
6. Centrifuge Filtration tube at 5,000 g for 15 min.^b)
7. Add 200 µl TE to Filtration tube to re-suspend the DNA on the filter with pipetting.
8. Transfer the ARP-labeled DNA solution to a 1.5 ml tube, and again add 200 µl of TE to Filtration tube to completely transfer the ARP-labeled DNA from the filter to the 1.5 ml tube.^c)
9. Store the ARP-labeled DNA solution at 0-5 ºC.

a) Ethanol precipitation can be used in place of Filtration tube to purify the ARP-labeled DNA. After ethanol precipitation, dissolve the DNA pellet in 100 µl TE, and determine the DNA concentration.
b) If the DNA solution still remains on the filter after centrifuging, centrifuge for another 5 min, and then proceed to Step 7.
c) The recovery rate of DNA using Filtration tube is 90%, so the approximate concentration of ARP-labeled DNA is 2.25 µg/ml. For more accurate determination of the number of abasic sites in sample DNA, we recommend measuring the actual DNA concentration.

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 each concentration of the ARP-DNA standard solution.
3. Add 60 µl of the diluted ARP-labeled genomic DNA solution per well. Use 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:
Preparation of Solutions for Day 2
Washing buffer solution: Dissolve the contents of the Washing buffer packet in 1 L of deionized or distilled water. Store this Washing Buffer solution at room temperature. HRP-Streptavidin solution: Dilute HRP-streptavidin with Washing buffer solution to prepare HRP-streptavidin working solution.
5. Discard the DNA binding solution from all the wells, and wash the wells five times with 250 µl of Washing buffer solution. After discarding the Washing buffer solution, invert the plate and tap it on a paper towel several times to completely remove the solution.
6. Add 150 µl of diluted HRP-Streptavidin working solution to each well, and incubate the plate at 37 ºC for 1 hour.
7. Discard the solution in all wells, and wash the well five times with 250 µl of Washing buffer solution.
8. Add 100 µl Substrate solution to each well, and incubate at 37 ºC for 1 hour.
9. Measure the O.D. at 650 nm and prepare a calibration curve using the data obtained from the ARP-DNA standard solution wells.
10. Determine the number of abasic sites in the genomic DNA using the calibration curve.

Typical Calibration Curve Prepared by ARP-DNA Standard Solutions

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 HRP-Streptavidin activity.
　
Assay procedure
1. ARP reaction with isolated genomic DNA
2. Purification of ARP-labeled DNA with a Filtration Tube
3. Binding of the ARP-labeled genomic DNA on a microplate
4. HRP-avidin treatment
5. Coloring reaction with Substrate Solution
6. Measurement of O.D. at 650nm

Storage condition
This kit is stable for 12 months at 5 ºC.

Reviw
"Oxidative Stress, DNA Damage and Human Diseases" is available.