Fourth Annual DNA Grantees' Workshop
Tuesday, June 24, 2003
Research Update Briefings: Ongoing Projects (Continued)
David Coffman, Moderator
Biography
Innovative Hybridization DNA Typing for Forensic Applications
Winston C.H. Chen
Biography
MR. COFFMAN: Our first speaker is Dr. Winston Chen. He received his degree in chemical physics from the University of Chicago. He currently works with the Photo Physics Group at the Oak Ridge National Laboratory, and he's going to be talking to us today about innovative hybridization DNA typing for forensic applications. Dr. Chen.
DR. CHEN: Good afternoon. Today I'm presenting something to you that's really different from all of the previous talks. Why? Because it's not working yet. These are some new ideas on hybridization detection for DNA typing in the interest of forensic applications. Chen: Slide 1
I'd like to acknowledge my collaborators: Professor Bruce McCord from Ohio State University and Dr. Eric Buel from Vermont Forensic Laboratories. We are trying to see if we can take advantage of the computer industry. As everybody knows, people raised billions and billions of dollars on developing the computer and the personal computer (PC), so we'd like to see whether we can use a PC for DNA detection. If we can, obviously that would make the detection much cheaper and more convenient and portable.
Our approach involves a floppy diskette and a floppy drive. We want to use the floppy diskette as a DNA chip and use the floppy drive as a detector, so then any hybridization can be detected. Chen: Slide 2
How do you do that? As you all know, a floppy drive detects a magnetic field. Any change of the magnetic field will cause the possibility of the floppy drive to do the detections. As you all know, people use the biotin and straptavidin reaction, and that straptavidin can be coated on a magnetic bead. With that, then there's a possibility that we can use the floppy drive for DNA detection.
If this works, then this should be very, very high-throughput. As you all know, one 3-inch floppy drive can hold 1.4 megabytes of data. That means you can have 1.4 mega-hybridization sites on one diskette, on one chip.
Most hybridization detection or DNA testing is based on labeling, like dye labeling or radioactivity labeling. Instead, we use a different kind of labeling, one that involves magnetic particles for the floppy drive detections. I want to emphasize the particle. Let's say that one DNA molecule attaches to one dye molecule. In this case, the detection sensitivity is limited by that one dye molecule and the typical sensitivity is in the range of a fentamole. In principle, that particle can be any size you want, and if the particle is big enough, then the sensitivity can go down to one hybridization or one duplex. Certainly we don't aim for that at this moment, but there's a possibility to make the sensitivity much higher. Chen: Slide 3
You may ask why I'm working on this kind of thing. Hybridization detection is available from Alpha Matrix and many, many other companies making different types of approaches. Nevertheless, they are very expensive and are usually in the price range of $100,000. As you all know now, a computer, if you just want to use a floppy drive, is probably less than $2,000. The key incentive, then, is cheap, cheap, cheap. Chen: Slide 4
Second, most people here probably brought their own computer, so it's certainly portable.
Third, I just mentioned there's a potential of really sensitive detections, so if it can go down to a really low level, then there's a possibility that PCR (polymerase chain reaction) may not be needed any more.
A floppy drive's output is usually digital, either a 1 or a 0. In the laboratory, instead of getting a data card of just 1 and 0, we can get an analog output, and it can be displayed on a PC, which will give us some kind of quantitative idea about hybridization. Chen: Slide 5
How do we do it? The substrates of a typical floppy diskette are usually Mylar—it is called PET (polyethylene teraphthalate)—and have a layer of 125 microns and a magnetic material, typically iron oxide. Chen: Slides 6 and 7
We try to use laser ablation or some kind of chemical to wash out these coatings so that we can try to put the DNA onto it. Then this DNA will be ready for hybridization, and then there is some more chemistry for how to do this.
In thermal particle attachment, let's say that we have the probe DNA, the target DNA, and the hybridized DNA. Then we attach the magnetic particle on top of the target DNA, for which the magnetic particle will be used for signal detection. We also found that when the target DNA is already attached to the magnetic particle, the results are similar. We also can do a lot with the detection. Chen: Slide 8
Well, we tried to produce the magnetic particle ourselves by laser ablation. Then we tried to target the DNA with the magnetic particle. Chen: Slide 9
This is just one example with a laser ablation in a solution of iron oxide, and then with another laser we can see the formation of particles by the heating and [inaudible] laser scattering. This one is without iron oxide. Chen: Slide 10
After we produced the particle, suspended it in isopropyl alcohol, and put amino triethylene in there for chemical reactions, we then tried to attach DNA to the magnetic particles. Because we wanted to see whether we can use the floppy diskette to detect the magnetic particle, next we tried to demonstrate the magnetic particle or the damaged disk. Chen: Slide 11
We have two spots. Spot 1 somehow disappeared already. We used the laser ablation to clean up the magnetic layers and then with the DNA attached to the magnetic particle, we can repair it. By using the computer check, we can find the error number and reduce that down to close to zero. This indicates that the magnetic particle we produced can be used for a repair of the diskette. Chen: Slide 12
Can we do the hybridization on the diskette? For this, we used the so-called beacon DNA as target. This beacon DNA has the properties that the luminophor and the quencher are very close to each other. When they are close to each other, there is a quenching process, so it's not fluorescent. But when it's hybridized, they're far apart. Then you will see a fluorescence because of the hybridization. Chen: Slide 13
On spot B, we can clearly see the fluorescence. This indicates we can do the hybridization on a floppy diskette. Chen: Slide 14
The next question is can we use a disk reader as a detector? Chen: Slide 15
Yes, hybridization can be detected on a floppy diskette with a modified disk reader. Hybridization can also be detected on a floppy diskette using a scanner, but floppy drives are cheaper than scanners.
Anyway, we used an inexpensive Epson scanner and could easily detect one fentamole of the DNA with the particle attached to it. Chen: Slide 16
So that's all that I want to present. Now we can go to the conclusions. We see that we have reasonable progress in terms of developing the computer for detection. This session is supposed to be on model technology. Unfortunately, I have to tell you that if you try to buy this technology tomorrow, it's certainly not available. Chen: Slide 17
Unfortunately, we tried to take advantage of well-established computer technology, but the diskettes from the computer industry—to our big surprise—are very different, and the substrates are very different, too. Therefore, we have some reproducibility problems.
It's not perfect yet, but I'm hoping that maybe 1 year from tomorrow this will become a real technology. The scanner seems to be working well. We're very excited about it, but I hope it's not the same as our diskette approach in that we were really excited about that too but then we ran into problems with the diskettes. Anyway, we are making progress and we hope that we can push this technology into a real forensic application in the near future.
I'd like to thank NIJ for its funding and thank you very much for your attention.