Fourth Annual DNA Grantees' Workshop

Tuesday, June 24, 2003

Question-and-Answer Session

DR. VOSBURGH: I know we're running just a little behind, but we'll take a couple of questions, Jan Sikorsky.

MR. SIKORSKY: I have a question for Dr. Ballantyne. Have you guys done any work to determine the efficiency of polymerase progressions through those photo lesions?

DR. BALLANTYNE: You mean the native polymerase, not the translation one. No, not in terms of efficiency. We haven't done any quantitative work. We're taking an empirical approach. By that I mean, is the profile gone or not and, if so, how does that correlate with the types of damage we see/ But we haven't done a lot of work on that.

MR. SIKORSKY: Would you be interested in doing some work on that?

DR. BALLANTYNE: Absolutely, yes.

DR. VOSBURGH: Questions? Okay, Roger [Kahn].

DR. KAHN: Jack, sometimes there was quite a bit of damage in your time zero with the alkaline gels. I was interested in that, because that means something about the preparation of the DNA presumably is causing damage itself. Additionally, if nicks stop polymerase or creates incomplete chains, was there anything you observed in terms of preparation or storage or anything that prevented that?

DR. BALLANTYNE: No. In fact, we're working on this exact topic. The preparation of DNA damages DNA; that's well known. We have found that single-strand breaks are caused in dehydrated DNA. This is unpublished work, but after speaking to my Ph.D. student about recent data, today actually, we're sure that single-strand breaks do occur as a result of dehydration.

The problem is that we're measuring oxidative damage. It's well known in the literature that oxidative damage takes place as you process DNA. It's an interesting phenomenon.

In fact, we think that some of the damage that will affect forensic stains may be repairable at that early stage. We may have to pretreat the DNA with a repair system first, especially single-strand gap repair, and a DNA polymerase type with ligation, then do the analysis. If single-strand breaks are sufficiently frequent, when you start manipulating the DNA and especially if you run it through a PCR, then that will cause the loss of profile typing.

So we're very aware of the fact that manipulating DNA can cause a certain amount of damage. Obviously, the only time this would be used is in those situations where you have a really difficult situation, and you don't want to exacerbate any damage to that by your own manipulations. So we're quite well aware of it, and we're just trying to do some fundamental work to find out precisely what is taking place apart from the oxidative damage. As I said, we have some preliminary data that point to the single-strand breaks.

DR. SENSABAUGH: I'll speak to Jack on this: If it's oxidation, as you think it is, then extracting with oxidation traps would be a solution.

DR. VOSBURGH: Do we have another question?

DR. McCORD: I just have a quick question. How long would that blood stain have to sit out under normal sunlight to approximate the amount of flux of UV radiation that you've used in your experiments?

DR. BALLANTYNE: Again, you have to do it empirically, because the literature is not very clear on this. Basically, you measure the flux that you would have on, let's say, a summer day in Orlando. The 102 hours that we see with a stain with UVC is probably equivalent to several years of exposure.

But remember, this is exposure in the absence of the other factors, like heat and humidity, which we firmly believe in. We all know as forensic scientists, it's the combination of all of them. The next phase of our work is to do a more definitive controlled set of experiments with known humidity and heat because obviously sunlight contains infrared radiation.

We realize that these are test systems and we're using them initially as a proof of concept. Can we detect the damage and show how physiologically relevant they would be, for example, in sunlight over time?

VOICE: You're talking about single-strand damage or something like that on the specimens. But even before that, there's bacteria damage of these samples other than single-strand breaks. So how do you differentiate that part of it? Bacteria degradation is much more of a problem compared to the single-strand breaks.

DR. BALLANTYNE: We just take a biochemical approach to this, whether it is caused by bacteria or whatever. You either have single-strand breaks, double-strand breaks, or all these types of damage. Often the bacterial effects will be double-strand breaks. I would just treat it as a double-strand break. Well, how do we repair that? Well, we may not be able to repair it.

The eventual aim—and we've never put this in any proposal—is to repair double-strand breaks, and that's very, very difficult. If one could do that, that would solve a lot of problems. We've got some ideas, but that's really the goal that I don't think too many people would suggest could ever happen. However, there may be a combination of methods that can do so. But I think people like ourselves would be willing to try that.

DR. VOSBURGH: Okay, we're going to take a 10-minute break to get our computer set up and thank our guests.