I developed the ddTUNEL assay to examine modification of DNA bases for assaying chemotheraputics.

ddTUNEL is just like TUNEL, but we use ddUTP instead of dUTP. This gives a 3'H end cap on every 3'OH DNA end. We tend to use biotinylated ddUTP and visualize with labeled avidin.

One can examine DNAse type II cuts by then incubating with alkaline phosphotase and then rerunning ddTUNEL, which labels 3'PO4 ends (which are converted to 3'OH by the claf intestinal alkaine phosphatase; CAIP-ddTUNEL.

If you have capped all the 3'OH and 3'PO4 ends with CAIP-ddTUNEL and added avidin/streptavidin, then one can convert all the damaged DNA bases to new 3'OH ends with a combination of a DNA repair enzyme and CIAP. I use the E. Coli enzyme Formamidopyrimidine-DNA Glycosylase (Fpg or Mut M); Fpg-ddTUNEL.

Firstly you inclubate you fixed, permeabilized cells with CAIP, wash and run ddTUNEL, then visualize half the samples with , say Texas Red Avidin, and the other half with unlabeled avidin. Blot with biotin and then wash, incubate with Fpg, then wash and a second incubation with CAIP, wash and run ddTUNEL and visualize these second cells with Texas Red Avidin.

The ddTUNEL/CAIP-ddTUNEL gives you total 3'OH/3'PO4 ends (including AP converted to 3'PO4 by AP-Lyase) and Fpg-TUNEL gives modified pyrimidine's and AP sites. You have ONE biotin for each 3'OH end that you label, regardless of source.

Here is our Biotechniques paper

http://www.biotechniques.com/multimedia/archive/00100/BTN_A_000113439_O_100375a.pdf

I'm not really asking about ways to detect DNA damage, but rather how to keep MG from directly damaging DNA, while still allowing it to generate ROS/oxidative stress. And on the other hand, keep it from inducing oxidative stress, but still allow it to directly damage DNA.

It's a quite difficult task, since usually there is a direct relationship between ROS generation and DNA damage. However ROS elevation can act not only through direct DNA strand break induction, but by induction of nuclease activities. So it would be interesting to see whether inhibition of nuclease activities can uncouple these two processes in your system.

I'm curious about your reaction conditions and method of exposure. You indicate you are using 'mustard gas' so it's not clear to me if you are actually gasing a solution, gasing a dried DNA preparation, or as is most typical, introducing a nitrogen mustard compound (presumably, HN2, (bis(2-chloroethyl)methylamine) to a DNA solution at some specific concentration by delivery from a stock solution in an organic solvent. Obviously, the experimental conditions will determine the set -up. However, assuming you are exposing aqueous solutions of DNA to the nitrogen mustard compound, you can use a combination of incorporating various types and concentrations of known radical ion scavenging agents with known scavenging capacities to your reaction buffer prior to HN2 exposure (i.e. DMSO, EtOH, etc.), and/or using oligos of known sequcence as your DNA damage targets. Use of the radical scavengers should allow you to differentiate between indirect damage due to ROS production and direct damage due to direct interactions between the HN2 and the DNA target. Avoid the use of radical scavenging agents that possess nucelophilic groups such as amines, since these agents may react with the HN2 directly and reduce its effective concentration during the reaction (ie. Tris-Hcl, glutathione, etc.), thus causing artifacual and inconsistent results. Use of oligos of known sequence as your DNA damage target offer the obvious approach of measuring the HN2-specific damage of interstrand cross links formed at appropriately designed sequnces within the target duplex. Such lesions could be detected by, for example, disruption of an appropriate restriction enzyme cleavage site and visulaized by autoradiography of the 32P labeled oligo. This approach also offers the advantage of precise quatification of damage yields through a combination of scintialtion counting and extablishing the proportional yields of the different forms of product (undamaged vs. damaged) via digital densitometry.

I hope this was helpful, and good luck!

What assay do you use for ROS detection?

I don't have one yet. I'm thinking ahead right now.

You may use several ROS inhibitors to prevent its ROS related activities. DNA damage can be because of ROS. If you prevent production of ROS, you might inhibit the ROS-mediated DNA damage. You can find some of the inhibitors from one of my paper.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0047427

Thanks Bhupesh, I'll look those up.