Fourth Annual DNA Grantees' Workshop
Wednesday, June 25, 2003
AFTERNOON SESSION
Use of Robotics for DNA Isolation and Quantitation
Jeffrey B. Ban
Biography
MS. BASHINSKI: Our next speaker is Jeff Ban. You know him from Virginia. He has worked for several years in Broward County [Florida] before he went to the Virginia system. He was in charge of the databank and CODIS part of the Virginia system, but since 1993, he has been the section chief of the DNA unit.
MR. BAN: Let me start out by sort of following what Julie said. Lois asked myself and the rest of the panel to share with you some of the things that are going on in our laboratories as well as some of the bottlenecks that we see. Virginia currently is using robotics to try to speed up the process. We've heard a number of speakers talk, and I think most laboratories can agree, that there's only so many spots to put new examiners, plus there's not a lot of examiners out there to hire at this particular moment. Ban: Slide 1
One of the things that we looked at in Virginia was moving toward robotics in order to do faster, quicker, and hopefully more precise DNA cases. We currently are working with Beckman Coulter's Biomek 2000 robot, and we're also using the DNA IQ system to isolate the DNA and the AluQuant Human Quantitation System from Promega for the quantitation process. Ban: Slide 2
Right now we've been using the system in our headquarters in Richmond. Since last July [2002], we've analyzed almost 2,500 samples and about 251 plate controls. I'll explain what that is in a minute. Ban: Slide 3
When Pat [Wojtkiewicz] gave his presentation yesterday, he talked about contamination, which was one of the things that we were concerned about as we got into the arena of using robotics. In fact, when we first started working with this, Promega came out to set up the robot and Beckman Coulter came out to set up the initial program. Susan Greenspoon, who did most of this work in my laboratory, and I noticed that the platform was shaking, and we looked at each other and smiled and said that there is no way that we're going to let this thing throw DNA all over the place. Through a lot of validation and modifications and through working with Promega during the past 18 months, the system has become pretty much perfected.
We are just about ready to go online in our northern laboratory, which is up here in Fairfax. Our western lab out in west Roanoke, and our eastern laboratory in Norfolk will probably be online in the next week or two.
We initially started out by using all types of case evidence where enough material was available in case we had to go back and reanalyze the sample again. The idea was that if the robot had a hiccup or something or there was some type of contamination, you could still go back and reanalyze a sample without compromising the case. In situations where we didn't have a lot of sample—in other words, it was a one-shot deal—we continued to do them manually. Ban: Slide 4
As I indicated, we went online in July of last year [2002], and about 3 months later, my staff indicated that most of the samples were one-shot deals. That is, a lot of the samples were from touch cases, such as carjackings, where investigators swabbed the steering wheel or collected a cigarette butt. They're low-level samples that don't make good use of the system's efficiency.
So we sort of changed our policy: We would move forward with this even if it's a one-shot deal or if it's a crime against property and not a crime against an individual.
We started off the program pretty slow, allowing everybody to run a couple of cases at a time on the robot and to do their extractions manually until they got to the point where they were really comfortable with this. I have to say now that the individuals who routinely use this can't even imagine going back and doing extractions manually, and we routinely take cases that are, like I said, touch cases, such as steering wheels and things of that nature. Those—the one-shot deals—are run on the robot. The examiners are extremely happy.
We did a lot of validation up front. Pat [Wojtkiewicz] was talking yesterday about using the robotics in his laboratory system, particularly that it has a cap on the tube and it opens up the cap from sample to sample. In this particular platform, you have a deep-well 96-well plate. It's probably about that high [indicating], and all of the caps are open. So when you initially look at this, you wonder about the chances of not getting contamination, so we routinely run a number of different controls.
This is just to give you an idea of the setup for a 96-well plate. The first two rows, for instance, should be sort of an orangish color. One examiner could load his/her samples into this first series of rows, for example E2. We call the blank row a plate blank, and then another examiner could load his/her samples in the next row. Ban: Slides 5 and 6
You'll notice that samples from different examiners are not set directly next to each other. It's set up so that the last well run by an examiner is a plate blank. Examiners do that through the entire process.
We have run 251 of these plate blanks, and we're keeping track of how many times we have seen contamination in there. One time we've seen a light amount of contamination; however, we're not sure that we can even attribute that to the robot and that wasn't a manual situation.
In this situation here, where you've got four different examiners running their samples, and this is probably about 1 hour and 55 minutes. The system allows you to modify the program depending on how many samples you're running: You can run an 88-sample method, which would be pretty much the whole plate, or you can run a 48 or 24. So if only one examiner is running samples that day, you can initiate the program to run only 16 wells.
The arrows point to the plate blank that was run by that particular examiner. We have four quality-control checks that we monitor.
If any one of those samples is between two examiners, then all the samples that are around that particular control would have to be rerun. But like I say, we've seen that only once out of nearly 2,500 samples.
So far, these are the types of samples that we have run on the robot with very good success. Like I said, these are low-level samples: earrings, toothpicks, blood swabs, cigarette butts. Ban: Slide 7
There have been a number of talks earlier in the program about different quantitation systems. Just to give you an idea, here is the AluQuant system. We took known amounts of DNA and looked at this during a 5-day period. We found a little bit more error at the higher end. In this particular situation where it was 4 nanograms, the average DNA concentration approached 3.25 nanograms per microliter. But in the lower area, which is most likely what you're dealing with, you'll notice that it's pretty good. Ban: Slide 8
The nice thing about this is a program called the AluQuant calculator. With your controls, you run the plot that your samples are compared against. So instead of getting an autoradiograph or a luminograph and having to eyeball it, the program pretty much tells you the exact concentration of N. Ban: Slide 9
That's pretty much it. These are the individuals in the laboratory. I mentioned Susan Greenspoon, who's done most of the work. These are the members who have helped set up the robot in other laboratories in the system. We've also worked closely with Promega Corporation, where Allan Tereba, Bob McLaren, and Dan Kephardt are helping us to get the program up and running. Ban: Slide 10
MS. BASHINSKI: Thanks, Jeff.
I have just one little question for you. Are you using barcoding or what kind of use are you making of barcoding in this process? Do you barcode your evidence that comes in initially?
MR. BAN: No, the evidence is not barcoded. What happens is, as I mentioned, the deep-well 96-well plate that's used is set up like the grid that I showed you up there. Plus, there's a worksheet next to the robot. Each examiner loads his/her own samples. Then, a technician who has been trained to run the robot puts the 96-well plates in the robot, initiates the program, and comes back in about 1 hour, and aliquots of your sample are in small amplification tubes. So at no time does the examiner or the technician ever handle a particular sample.
MS. BASHINSKI: One other thing: With sexual assault evidence, do you do differential before using the robot?
MR. BAN: Yes. Yes. Dr. [John] Herr's group talked earlier today about trying to come up with some type of bead technology for capturing the sperm, and the problem right now is that almost all samples except for sexual assault samples can be run from the very beginning. You cut the sample, put the chemicals in the tube, and load it onto the robot.
For sexual assault type of samples that require a differential, we have to do that up front, which takes about 2 hours, and then everything from that point on is loaded onto the robot.
MS. BASHINSKI: Thanks.
DR. KAHN: Just a quick one, Jeff: With the cut-ins, do you just drop the material directly in the tube, or do you have to liquefy or suspend the material first?
MR. BAN: There are two ways that you can do the DNA IQ procedure: It can be done manually or in the robot. Initially, you start out the same way for the robot as you would if you were doing it manually. It's a very quick process: You put the chemicals in the tube, let it soak for about 30 minutes, put it in the heat block, pull out the sample, spin it down so you get your supernatant, and then throw that on the robot. From that point on, the robot does everything else.
MS. BASHINSKI: Thanks.