All right. Thank you, ladies and gentlemen. All right. Dr. Cotton, would you resume the witness stand, please.
Actually, perhaps the witness could stay there. That's where I was going to direct her next.
All right. Great. Good afternoon, Dr. Cotton. You're reminded you are still under oath. Mr. Clarke, you may proceed.
Now, Dr. Cotton, this process of electrophoresis, and are we talking about what is in basically step no. 3 on the large--the RFLP method diagram?
And you've described the fact that through the use of electrophoresis, these fragments that differ in size basically go to a different portion of the gel; is that right?
And, your Honor, if I may, I'd like to utilize a previous chart which has been labeled People's exhibit 215.
And in particular, Dr. Cotton, I'd like you to assume there's been previous testimony about the use of this chart in a description of the use of electrophoresis in typing of proteins.
First of all--and I'd like to direct you specifically to what's labeled no. 3, load gel in the photograph above that. Does that appear to be a gel?
All right. Is that the same, different or similar from the gels that you're describing as part of the RFLP process?
Well, I'm sure it's different. I'm not exactly sure what kind of gel that is. If it was the RFLP process--and that looks to me like a glass plate although I can't tell that well. If it was the RFLP process, it would be a plastic tray that has sort of like boring something into a square or rectangular cake pan. But the edges of the tray are maybe about three-quarters of an inch high.
Okay. Then taking you on to step 4 on this diagram, People's exhibit 215, that's labeled, "4, electrophoresis," is that the same or similar to the electrophoresis process you've described in the RFLP method?
The tank that's shown in photograph 4 doesn't look very different from the type of tank we use. It has space at either end where buffer or some kind of a wick goes. It has a plastic lid that comes down to protect the user. What's really different here is that if you look at photograph no. 3 where it says "load gel," first of all, the position where the samples are loaded on an RFLP gel is very close to the top, more like what I've drawn in this diagram where's there, it's maybe quarter of a way down the gel. And these are--in the RFLP procedure, these are liquid samples that are loaded using the pipetter with a disposal plastic tip.
Okay. And then lastly on this exhibit 215 where it's labeled, "running the gel," do you in fact run gels during the RFLP method typing process?
Yes. You--it shows there a series of gels and two power supplies up on a shelf and we have a similar looking power supply that's connected to the gel tank such that you have a negative and a positive electrode which is creating the current, electric current which is pushing these DNA fragments along through the gel.
Your Honor, with the Court's permission, could 215 just be brought over to this side so the remaining jurors can have a look at it?
Now, Dr. Cotton, at the time the electrophoresis or step 3 on People's exhibit 242 is completed, can you see these different fragments on the gel?
The next step in the process is to create a permanent record of the separation of the DNA fragments in the gel. If you try to pick up a agarose gel with your hands, it would be like trying to pick up a quarter inch slab of Jell-O. It would break apart in your hands. So whereas all the information you want is in this gel, you have to have a permanent way of storing this information. Can I backtrack for one second--
Let's still consider this two chromosome or the two DNA pieces from the diagram a few places back. Let me make sure that I straighten one thing out and then go on. I talked about a fragment of DNA that was nine base pairs and another fragment that was 15. Let's put this more into reality for this test. The sizes we might look at, you could look at a size that was 900 base pairs and a size that was 1500 base pairs. This is more like the size range. And actually you can look considerably larger than this. But this is more like the size range of pieces that you would be looking at in this test. Still, do where the differences in size, we do to the different number of repeats in that section of DNA. So that if we go back, we have our 900 base pair piece has moved further down through the gel and the 1500 base pair piece has not moved as far.
When you have this separation in the gel, this is the information that you really want. And the entire rest of the procedure are the set of steps that allow you to visualize where these pieces of DNA went. So if you understand that where the DNA came to in the gel is rel--is a measure, an indirect measure of its size. That is, this distance traveled is an indirect measure of the size; the further the distance traveled, the smaller the piece of DNA. The shorter distance traveled, the larger the size is. This is the information you're after, and the entire rest of the procedure is how you see this visually.
All right. And, your Honor, for the record, the witness has been referring and writing "further" on People's exhibit 244.
All right. Dr. Cotton, then would you be or are we proceeding to step 4 on the diagram, the RFLP method?
Yes. And I won't actually--maybe I won't draw another one because this is pretty nice here on step 4. What you want to do now is keep this record or make a permanent record of where these DNA fragments are. And the way that's done is to transfer the DNA in the same relative positions from the gel onto a nylon membrane. And that step or that process is illustrated in step 4.
Or a pointer. If you can reach--I think we've got a pointer right at the entrance to the--in fact, Dr. Cotton, did we or did you bring one of these membranes to court today?
All right, your Honor. I would ask to have marked as People's next in order, I believe 245?
Dr. Cotton, if I can show you what seems to be an object about the size of what, eight inches by eight inches or so?
Okay. That sounds like a more precise description. What is--and referring to what will be marked People's exhibit 245, what is that?
This is how the membrane comes to us. It's in these red pieces of--this is just a piece of paper and it has written on it MSI, and that's the manufacturer of the membrane that we use; and sandwiched in between the two pieces of paper is a nylon membrane. Now, if I were doing this in the lab, I wouldn't be touching it with my fingers. You would be handling it with gloves because fingerprints wouldn't be helpful. Any of the oil from your fingers is really the problem. And this hasn't been used in the lab, so it's clean. And this is the nylon membrane.
So in the example here, you have a sponge. And in our lab, we use something that's called a wick. But it's all going to do the same thing, so even though this is slightly different from my lab, I'm just simply going to stick with this. And then on top of the--and the sponge is soaked with some solution, and on top of the sponge, you place the gel. So you can see the gel here is green and you have a green layer here. That's the gel. The gel isn't really green. It's really just kind of white. And you can see that the membrane isn't really orange, but they made it orange. And--anyway, so you have that gel, and you simply--I had a gel here. I'll use the top of this thing. You would very carefully take this nylon membrane and lay it down--
Actually--I'm sorry, Dr. Cotton. Perhaps you could demonstrate that on the actual bar in front of the jury so that more can see.
I had my gel sitting right here. I would carefully lay this nylon membrane down on top of the gel and let it fall down easily. And it--it would be wet beforehand and the gel is damp. And then you can--when I did this in research, I used my fingers, but in the lab, we now use a pipette. But you would smooth down the nylon membrane so that it was in easy close contact with the gel.
Yes, sir. Thank you. Now, what's the purpose of this is to have the DNA move out of the gel and come in contact with the nylon membrane. So if you go back to the diagram here, the salt solution that's in the sponge will move up through--oh, well, wait. Let me--let me finish the order here. We have the sponge, the gel, the membrane, a big stack of paper towels and a weight. So the idea is that the solution moves up that's soaking in the sponge, moves up through the gel and carries with it the DNA and it's sucked up--the solution is sucked up into the paper towels, but the DNA becomes trapped on the nylon membrane. So if you were to go back to this diagram, the membrane would be layered right on top of the gel and the DNA would move up through the gel and become attached to the nylon membrane. The paper towels and the weight serve the obvious purpose, the same as if you had a puddle on the counter and you put the paper towels on it. The solution would be soaked up onto the paper towel. It's not a very elegant contraption, but this is one of the most important procedures in all of molecular biology and it's used all over the world.
Mr. Clarke, the diagram that Dr. Cotton referred to again, the drawing, I believe that's the electrophoresis diagram?
Now, I have omitted one important feature here, and I--somehow I always have trouble getting this in the right order. Just before you set this setup up, you treat your gel so the DNA fragments that are double stranded will come apart and become single stranded. And you do this by soaking the gel in a solution of sodium hydroxide. So basically--and the other method that we talked about was heating it, but if you heat it, the gel will just melt. So you can't do that. So you're going to separate this what was double stranded to now be single stranded in the gel and then you're going to do this transfer so that as the DNA is bound to that nylon, it's in a single stranded form. And that's the important thing that I left out.
Actually two questions if I can. First of all, why doesn't the DNA travel through the membrane and up into the paper towels?
The DNA binds to the membrane and it becomes trapped there. The membrane doesn't have sufficiently large pores in it for the DNA to get through.
Okay. And then you also referred to this process as not being particularly elegant; is that right?
Well, it's very elegant, but it's not very fancy. It doesn't require a lot of equipment. You don't need any electricity. You could set it up in your kitchen if you really wanted to.
KEY QUOTEIs this particular step limited to forensic science or is it applicable or is it actually used in any technique involving use of the RFLP method?
It's used in any technique where RFLP is used and it has other uses as well. But if you go into any of the scientific journals, you probably can't go through a journal that talks about molecular biology or biochemistry without finding an article where this procedure is used. This procedure is called a southern blot. And it doesn't have that title--oh, yes, it does have the title here on the diagram where it says, "by a technique known as southern blotting." it's called a southern blot because the scientist who devised this procedure in 19--late 1970's, mid 1970's is--his name is Ed Southern and he's from England, and his last name became attached to the procedure because it's so widely used. So it's called southern blot. That's the name of the scientist who devised the procedure and it's been used since that time.
All right. So now is it correct then that this membrane that will be marked 235 in this process now has single stranded DNA attached to it?
Yes. And the important thing is, however the strands were separated in the gel, they are now--that is, however the DNA fragments, that is the large one versus the small one, were separated in the gel, that same spacial separation is now on the nylon membrane. So the DNA is attached to the same nylon mem--attached to the nylon membrane and the pieces are in the same relative positions that they were in the gel when the gel was turned off.
What's the next step in this RFLP typing process after you have the membrane with the DNA attached to it?
The next step--I have to read what's on the board for a second to see where I am on this.
Okay. So over here (Indicating), after the blot, you have this statement on the board which is correct; "the DNA fragments are now on the nylon membrane, but are not yet visible." now, actually it shows you as if something was visible, but really, when you put the nylon down and you take the nylon off the blot, it looks just plain just like you saw it. It doesn't--except it's wet. And now you get to the point of the next few steps that allow visualization. And here--here's where it becomes important that you understand that you can take DNA fragments apart and put them back together, because now you're going to take what's called the DNA probe, which is simply a piece of DNA that you've made in the laboratory and has a radioactive tag on it. It's single stranded. And if your repeat was CAT, your probe would be GTA.
So in my single-stranded DNA drawing, I didn't draw the repeat cat, but let's suppose this is our repeat. If this is our repeat, the DNA that's on the nylon is now single stranded. So you have this available to collect the second strand and you would have the other side available to collect a second strand. So you take the nylon membrane and put it in a plastic tub or you can put it in a tube-like thing. But I'm going to use the example for a tub because it's a little easier. We use in the lab a Tupperware container, a square one, in which it was just about the size of that nylon membrane. So the membrane would be flat in the bottom and it's--and then the membrane is covered with a salt solution. The radioactive DNA probe is added to that solution and that solution is let sit at a specific temperature overnight. So overnight being, you know, about 16 hours perhaps. And during this time, this radioactive probe DNA, which is simply--the probe is actually, for what we do in the lab, attached to C's and g's. During this time, this probe that you've added will zip up with any DNA on the membrane that it can appropriately zip up with. The radioactivity is something that you can follow. If you take your membrane, add your probe, let it go overnight, take your membrane out, rinse off the excess probe, if I held a Geiger counter up to it, I would get a few clicks, the Geiger counter being able to detect the radioactivity. But that's not accurate enough. So instead of a Geiger counter, you would then--if we can go back here, here's our membrane that now has some probe bound to it. To detect where that probe has bound, you lay an x-ray film over the membrane. You do this in the dark. You put it in a metal cassette that sort of looks like the two--like two book covers then close up together. And the radioactivity that's on the membrane will expose the silver grains on the x-ray film creating a dark spot or a line. It's really a line. And that is the image of where the probe bound to the DNA. So if we go way back--if we go back to this diagram, this was originally our gel. Now this pattern of DNA is on the membrane and now you've added a probe to it, and the probe has bound specifically--if we make the probe so it binds specifically to the cat repeat, that's what it's going to do. So the probe is bound specifically here and here (Indicating) and it's been washed off everywhere else because only in these two positions is there a piece of DNA that it can come back together with. And we laid a piece of x-ray film over this membrane and allowed it to expose. And on the x-ray film, a dark line will appear in these two relative positions. That dark line tells you where the DNA fragment went to in the gel when the gel separation of these pieces occurred.
Dr. Cotton, these probes then, are they specifically designed to look for that repeat sequence that you're searching for on the membrane itself?
And when they recognize the sequence that they're looking for, the probe then actually mates up or zips together with the DNA that's already on the membrane from this blotting procedure?
Is the probe then simply a means for you to be able to see where the fragment is on the gel that you're actually looking for?
Yes. The probe--this is your tracer. It's your eyes. It's your tag. It--it--it--and you sort--we talk about it like it was a thinking thing, but of course it's just a piece of DNA and this is just a chemical reaction where--that allows these two DNA strands that were single stranded to come together. So it's simply your--your way in the laboratory of identifying where these pieces of DNA went that had the repeat that matches to the probe.
Then is the use of this radioactivity and x-ray film so that you can actually look and see where these fragments are?
It's properly called an autoradiograph. In the lab, we call it--we refer to it as an autorad. Just simpler to say, and that's what most people would refer--that's the term most people would use.
All right. At this point, your Honor, I would like to have marked as People's next in order--
Yes. A single what I believe to be an autoradiograph that's been--a copy of which has been previously provided to counsel.
And is that typically what an x-ray or autoradiograph looks like following the use of this RFLP typing process?
And with the Court's permission, could Dr. Cotton simply hold that up so the jury can see it from a distance?
This is one of the film--well, this is a copy, but it's a--the copies are very good. They don't--so that if I--
All right. Dr. Cotton, if you would just hold it up at the moment, and we will return to this particular autoradiograph later. But is this typical of what such an x-ray looks like at the end of this RFLP procedure?
You have to subtract out the lettering here and the lettering down here (Indicating).
The lettering you're referring to at both the top and bottom of this particular exhibit, is that done to aid in knowing what sample is--which sample is which basically?
Okay. All right. Fine. Would that autoradiograph then represent what is labeled on the RFLP method chart at step 7 and then step 8?
Okay. So step 7 is describing the creation of this x-ray film in its early stages by laying it on top of the membrane?
All right. Very good. Perhaps I can take that back. And if you would, could you have a seat back on the witness stand?
Actually, with respect to the RFLP method, the large chart, could that also be brought down so that all of the jurors could have--can look at it?
"control" is usually some kind of sample that helps you to determine whether all the procedures that you just finished using worked as they should, and it usually is a sample that you know what the results from the control should look like. And if they look appropriately, then it gives you some information that your test was done appropriately.
Now, referring to this RFLP typing process, do you utilize controls as part of that testing?
Well, it depends on what kind of control you're using. But for this procedure, you are ultimately looking at that x-ray film and visualizing your result, and later on you might make some measurements. But if we just talk about looking at it for the time being, the controls have a very typical appearance, one that you are accustomed to seeing. So if they look as they should, then you would make a general first-impression assessment that the test and the procedures that you used had been done appropriately.
Generally if they haven't worked properly, you would either go back and repeat it if you had--if there was a possibility to do that, or if you couldn't repeat it, you would probably just say that your results are inconclusive, because if your controls haven't worked, that--there's some implication there that how you worked on the actual samples may not have worked either.
Now, this autorad in People's exhibit 246 represents an autorad from an RFLP test; is that right?
This autoradiograph, is it in fact preservable? In other words, is it in a form that, for instance, can be looked at at a later time or by other people?
As far as the RFLP typing results--and first of all, did your laboratory obtain RFLP typing results in this case?
Are those RFLP testing results reflected on autoradiographs as an example of which is People's exhibit 246?
Now, I'd like to discuss a different area with you, and it relates in particular to when you are reading results from an autoradiograph. First of all, do you do that?
It means to me that on the autoradiograph, the position of two DNA bands or possibly the positions of all the bands from one sample to another are so close as to be visually--that I couldn't distinguish one from the other.
From person to person, there's so much variation in the length of the fragments that have a repeat, that if you look at several genetic locations, and for each genetic location, two samples are the same, and you look at another, and they're the same there, and you look at another, and they're the same there, and you keep doing that, the more genetic locations at which two samples are the same, the higher the likelihood that the two samples are in fact from the same person.
All right. Your Honor, if I may return to People's exhibit 242, the large board, because I believe it has an item that I would like to ask the witness another question about. I put it away too quickly.
Dr. Cotton, referring you to what would be step 8 on this board, People's exhibit 242, does that particular depiction--would that help you in describing what you mean by "match"?
All right. Could you then go ahead and describe that? And if the pointer would help--
This diagram and the things that I drew on the chart are illustrating the kinds of results you would get when you looked at one location on one chromosome. So since you get a copy of--you get two copies of the chromosome, you get one piece of information that you inherited from your mother and the other from your father. And so if we look at this sample that would be basically on your left, you have two dark lines on the autorad indicating the two DNA bands that are from this sample. And then if you go on to the middle sample, you have two bands on this autorad from this middle sample. And then if you go to the third sample, you also have two bands. The middle sample and the sample on the left that I'm pointing to, the bands are in essentially the same position. So it is possible that sample no. 1 and this middle sample, sample no. 2 could be from the same person because they have DNA bands that are in the same position.
This third sample on the right, which also has two DNA bands, these bands are clearly on a--in a different position than either the first or the second sample. That tells you immediately that the DNA in this third sample must be from a person who is different from either of these first two. These first two could be from the same person, but this third one must be from a different person because the DNA fragments are in different positions, which means they're different sizes.
If you were comparing--suppose this was a known individual and these were two evidence samples. This known individual in the third lane over would be excluded as being a possible contributor to the two evidence samples in the right--in the left lane and in the middle lane.
This is done visually as a result of the autoradiograph produced at the end of the RFLP typing process?
It's done visually and it's also--that's followed up by a more sophisticated measurement. But in fact, the first thing that you do when you get your x-ray film off is you look at it and you look at the controls and you look at the samples on there and you make some immediate judgment about whether it appears that this--procedures worked okay and whether it appears that you have inclusions or exclusions, and based on those inclusions or exclusions, then you might do further testing or you might not.
Following this visual determination, do you then in some instances turn to another process basically to determine whether or not your visual opinion is a correct one as to a match?
All right. We'll return to that later. But what I would like to turn your attention to--
And, your Honor, I'm going to leave the chart because I don't want to have to ask to bring it up again.
--and that relates to the amounts of DNA that are in a sample. And I'd like to focus your attention again on this RFLP typing method. If there's no DNA in a particular sample, what do you see at the end of the process on the x-ray or autoradiograph?
If there's no human DNA in a particular sample, at the end, you don't see anything. The lane is blank. There are no bands. It's just empty.
For the procedure as it's used in laboratories for human identification testing, you're specifically looking at--you want to be looking at human DNA and the test is designed to only detect human DNA. And, therefore--if you were using it in a research lab, you could see other types of DNA. But for this purpose, you're only looking for human DNA. If you have no human DNA there, you won't see anything.
So in your laboratory, your interest is in human DNA as opposed to looking at, for instance, animal DNA or other types?
What if you have some DNA in a sample? Would you necessarily see results at the end?
If you had some DNA in a sample, you might see some results at the end. There are two measures of whether or not you might see results, and one is quantity and the other is quality.
If your DNA is in good condition--and let's just go back to the spool of thread analogy. If your DNA is unwound off the spool and it's all in one piece, that's good condition. It's like it was in a nice fresh cell that somebody just drew out of your arm. If your DNA is in good condition and you have enough of it, you will get a result. If your DNA is broken up in a random manner--and the proper term for that is that it may be degraded--you may or may not get a result. And when you think about DNA that's broken up or degraded, you have to think of it sort of as a continuum. It could be only slightly degraded, that is, it's only broken up a little bit, in which case it won't affect your result, you will get a result, or it can be very, very broken up in very, very small pieces. And if that was the case, you would not be able to get a result with this RFLP test.
Well, I wouldn't use the term "die" or "perish." it doesn't really fit. "degrade" is the right term, or if you went to make that simple, you could just say it's become broken up into many pieces. And that can happen as a result of environmental effects, and you can generally sort of assume that moisture and heat facilitates degradation; that is, it occurs more rapidly, and cold and dry conditions preserve DNA.
If DNA suffers this degrading process, degradation process, will that result in changing the types from the original type of sample to a new and different type as a result of the process of degradation?
So this process of degradation, can it change my DNA into looking like your DNA?
KEY QUOTETaking you back to this small amount of DNA, what if you had more DNA in a better condition let's say? Then what do you see?
If you have more DNA and it's in good condition, then you will see bands on your autoradiograph or your autorad; and each time you use a different probe, you'll see bands that that probe recognizes. So you can get a lot of information if you have a good amount of DNA and it's in good condition.
Where do you draw the line? In other words, how do you know there's enough DNA there to be able to make an opinion or render an opinion about results from a particular test?
Well, you can sort of go about it from two directions. You can measure how much human DNA you have before you do the test and you can run what's commonly referred to as a mini gel, which is a really small gel, about, oh, two inches by three inches and assess whether the DNA appears to be broken up. So you can measure its quantity and you can get an estimate of its quality, and you could do that before you did the test and then say, "well, it looks good. Let's do an RFLP test."
And that's simply a step to help you evaluate, that is to determine approximately how much DNA is in a sample before you test it?
Right. If you just made that a lot smaller and which would then represent your mini gel, you can stain that gel with a dye in which you get a pink smear that looks like these smears that are on this diagram for step 3. And if you had a smear like what's shown in step 3, you would look at that and you'd say, "well, gosh, I've got DNA sort of spread out all the way from the top of my mini gel down to the bottom. That's pretty degraded." and if it looked like that, you'd be sort of--you wouldn't be sure whether it would be good enough for RFLP or not. If you--if this diagram in step 3 had only shown the pink color at the top, that's sort of what it would look like if it was in good condition and you ran a mini gel. You're looking at a huge piece because you haven't--when you do this mini gel, you haven't cut it with your restriction enzyme yet. So actually if we could insert an extra step here to be 1-A so that you extracted your DNA and then you ran your little gel and then you made this judgment. If you had DNA that was just hung up at the top of the gel, that would say--you would say, "well, I have very good DNA here. I should be able to go on and do an RFLP test." so DNA is all spread out such that it's--looks relatively degraded, then you would have to make a judgment about whether or not you thought it would be good enough to do an RFLP test or you were going to do some other kind of test with it.
As far as these bands as you see them and referring to for instance step 8 on People's exhibit 242, those are fairly dark bands in that depiction, correct?
You've also held up one autoradiograph, People's exhibit 246, which also shows a number of bands that seem to be very dark; is that right?
First of all, Dr. Cotton, to your knowledge, will we return to autorad and actually show them in closer detail to the jury?
But just for the moment--and if you could hold that up. I don't know if you can hold it up without that by the background. Let's do that.
Dr. Cotton, again showing People's exhibit 246, that shows a number of very dark patterns; is that right?
Now, with regard to samples, for instance that you receive in your laboratory, will the darkness or the intensity of those bands vary?
The darker the band, that reflects you had more probe bound to it. More probe means that you had more DNA there. So if we just--we don't have anything on here that has very light bands. But if you had something that had very light bands, that would be an indication that you didn't have very much DNA. If you have bands that are dark, that means you have more DNA. Now, there's other ways you can make that--the length of time that you lay the x-ray film over the nylon, the longer you lay it on there, the darker everything becomes. But it will still be relative. A darker band generally has more DNA than a lighter band, and there's a few reasons why that--there's a few technical exceptions to that, but that's the general and correct generalization.
All right. Then if you would have a seat back on the witness stand. Is it the case then, Dr. Cotton, that with regard to samples with not that much DNA in them, but enough to produce a banding pattern, you may see bands that are faint?
It means that instead of a dark crisp line, you have a difference between where there's no band and where there's a band, but the level of darkness there that is the intensity is much less. It's light.
You're also looking at the position of the band. So whether it's dark or it's light, it's the position that you're interested in. It does affect--that is, you think about it in your interpretation because you're assessing, "I have a lot of DNA here, I don't have very much here," and you may use that information in your interpretation. But it's mainly the position that you're interested in looking at.
What about in terms of faint bands and whether or not you can see them? Does that play a role--I assume that plays a role in your interpretation?
Are there any policies in effect in the laboratory at interpreting, for instance, results that have faint bands?
I don't believe that we have a specific written policy. We do have a common practice in our lab in dealing with faint bands, and we do follow that practice generally whenever we encounter faint bands. It's not actually a written part of our procedure.
There are two parts to it. In the further analysis that we haven't really discussed yet, which involves a computer imaging step, there is a standard level of darkness that we ask that the computer see in order to call something a band. In addition to that, we have to be able to see it ourselves. That is, both the computer has to be able to see it and the analyst has to be able to see it. And if we really are struggling with whether or not a band is light, we will ask several people to come in and cover up the adjacent lanes and make an assessment as to whether all the people involved in doing the analysis of the test agree that they can see a particular band.
When you say, "cover up the adjacent lanes," is that to ensure that whoever is looking at it wouldn't be bias by the presence of bands in other samples?
That's right. If you're working on something or another Ph.D. staff or one of the analysts is working on it, if I'm not sure whether or not something is intense enough for me to use it further in the interpretation, I might lay a piece of cardboard down on either side and just say to one of my coworkers, "would you come in and look at this and tell me where you see bands in this lane?" we don't always do that, but that's generally what we do when they get quite light.
As far as this process of DNA, once it's degrading, does this degradation process ever leave two bands appearing that weren't in the original DNA in the sample?
So in other words, you either see the DNA from the original sample, or if the degradation is too great, you don't see anything at some point?
Well, you've just said what the extremes are. You--you may have an intermediate result where you have the DNA is somewhat degraded. And the effect that that has is, you tend to lose the bands that are larger. That is at the top of the gel. So you may end up with a partial set of bands. You don't see the ones that are large, but you see the ones that are smaller in size. And that can happen. What won't happen is that with degradation, you won't have a band at one size and then have it converted to now a new band at a different size. That's not going to happen.
Okay. And perhaps lastly for today, with regard to this computer imaging process that you just described, can you tell us more about what that is?
It's sort of like this elmo machine actually. Well, kind of. The computer imaging system consists of a light box and on which you lay your film, a camera at the top and a computer monitor so that the camera is capturing the image of the autorad and displaying it on the computer screen for you, and then you're asking the computer to help you do some manipulations in terms of saying here's a band and here's a band and here's a band, and then the computer will also, because of its software, do a calculation for you that says, "the band in this position is approximately 5,000 base pairs."
Now, when you say the "autorad" that you have the camera actually focused on, is that an autorad just like People's exhibit 246?
And then this camera and this software in combination then review the results on that particular autorad?
Well, they--reviewing makes it sound like it's thinking about it, and it's not. What it's doing, it's visualizing--it's a video imaging system. It's taking a picture of that autorad and displaying it on the computer monitor, and then you are directing the machine to do particular things, not to the film itself, but to the image.
You're telling it where the control lanes are, you're telling it where the sample lanes are and you're telling it which lanes you want it to do a calculation, to find a band. That is, the computer will basically scan down, and where there's a dark area, it will identify that as a band, and where there's--so it's looking for changes in levels of darkness. So if it's clear and then it sees a dark area, then it identifies that as a band and then it sort of scans down and finds another band and identifies that. So it's making identification of a band position. And it can do this correctly and it can do it wrong, and the operator has to help it along. And then once you're happy that it's--that it's identified bands and you agree that those are bands, it will do this calculation for you that will give you an estimate of the size of that DNA and base pairs.
And that's the purpose of this imaging process, to assist you in interpreting results; is that right?
And also to establish the sizes for the various bands according to the methods that you just described?
Well, it's very elegant, but it's not very fancy. It doesn't require a lot of equipment. You don't need any electricity. You could set it up in your kitchen if you really wanted to.
No. It will not.
So this process of degradation, can it change my DNA into looking like your DNA?
It's your tracer. It's your eyes. It's your tag. It—it—it—and you sort—we talk about it like it was a thinking thing, but of course it's just a piece of DNA and this is just a chemical reaction.
We use in the lab a Tupperware container, a square one, in which it was just about the size of that nylon membrane.