Thank you, ladies and gentlemen. Please be seated. And Dr. Cotton, would you resume the witness stand, please.
Robin Cotton, the witness on the stand at the time of the evening recess, resumed the stand and testified further as follows:
You are reminded, ma'am, you are still under oath. And Mr. Clarke, you may continue with your direct examination.
Thank you, your Honor. Good morning, ladies and gentlemen.
THE JURY: Good morning.
DIRECT EXAMINATION (RESUMED) BY MR. CLARKE
Dr. Cotton, as far as these methods of DNA typing, and you have talked in broad terms about PCR and in more detail about RFLP, are these techniques simply a better way to type stains than for instance, ABO typing or conventional serology?
Yes, they generally will offer more information than conventional serology will offer.
Again we spoke about RFLP or you spoke about RFLP typing yesterday. The second technology that you mentioned briefly yesterday I believe you referred to as PCR; is that right?
It is actually about--it came along at about the same time, although it was not being used quite as widely early on as the RFLP process.
Are you familiar with who was the first scientist to use this PCR process in forensics?
As far as this PCR process--and many of us, not all of us have heard of the movie Jurassic Park--what does PCR have to do with that?
PCR is a way of saying I want to replicate a small section of DNA and it allows you to replicate a small section of DNA in a way that if you have a very small amount of starting material, you can go through sufficient number of replications so that by the end of the process you will have enough of this small section to then do some analysis on.
KEY QUOTEWhat role does it play, for instance, in old samples? And let's first of all ask can this process be used on samples of DNA that are perhaps years or decades or centuries old?
There are samples from, I don't know what the proper term would be, but very ancient, for example, bone samples that have had very short sections, copied using PCR to look at how genes have changed over time. That is, if you can have a sample from a human being who is several thousand years old and look at a particular sequence of the DNA, you might say, well, how does that sequence compare to the same sequence in--in modern human beings?
As far as this PCR process, is it like RFLP in the sense that it is a testing method or is it something a little bit different?
PCR is simply the process that allows you to make a lot of copies of a particular section of DNA and then the rest of the analysis may have many different forms. That is, what you do now with your section of DNA that you've replicated many times may not be the same for every purpose or every question.
Would it be correct then that PCR would be more like one step in a typing process when DNA is being typed?
Umm, I had been to several meetings where PCR was discussed. I had done some reading where PCR was discussed. My own hands-on experience came from visiting two different laboratories where they were actively doing PCR and learning from the staff in those laboratories how to do PCR, and these--both of these visits to other laboratories, one was two weeks and one was a week. I had an opportunity then to learn from the staff there to do it myself and then back--going back to cellmark to help the rest of the cellmark staff who also had--some of whom had also been out to get training, which was different than mine, to set up the PCR that we do there.
I went to a laboratory at Houston that was Dr. Tom Caskey and I went to Dr. Alex Jeffries' lab in the UK.
How about your own personal hands-on experience with PCR? Could you describe that, please.
Well, I had those three weeks and then I had some experience hands-on time doing the PCR reaction while I was--while we were in the process of doing validation studies to set up PCR in our laboratory, and a number of other people did, too. I don't--I am not doing the PCR tests now. The PCR tests are done by the regular staff.
As far as this copying process, is there a term used to describe what you actually do in taking small segments of DNA and copying them over and over?
Yes. The term is amplification. So people will talk about I have amplified a certain section of DNA.
This process--and I believe you said that this process of copying over and over is directed at short segments of DNA?
The segments that are used for forensic analysis are relatively short and I'm talking about perhaps a hundred, 200, up to 400 base pairs in length. That is a pretty--in DNA terms that is a pretty small piece.
So you are not talking about copying over and over this entire segment of DNA that you described as containing what was it, six billion bases or so?
No. You are not copying the whole thing; you are just copying a particular section.
With regard to this copying possess, would it help to use a chart or a pad so that you could draw what this amplification or copying process involves?
Your Honor, then with the Court's permission Mr. Fairtlough will I believe set up the drawing pad for the witness.
And Mr. Clarke, I noticed yesterday that you were using the dry erase type marker. Does that have a longevity to it, permanence to it?
All right. Your Honor, may this diagram be marked as People's next in order, which I believe is 247?
All right. Dr. Cotton, if you could step down from the witness stand, and what would be an appropriate label for this chart to aid you in describing this copying process or amplification?
All right. Now, if you would go ahead and if you could describe for the jury, please, what is this copying process?
Okay. Keep in mind now that you are starting--you have a sample and you have extracted the DNA, so those steps are the same as or similar to the RFLP process, and so let's just say for simplicity purposes that we have a single chromosome's worth of DNA that I'm going to use as a reference point here. So we are going to put in the tube our starting DNA and we are going to put in the tube a supply of the four DNA components; A's, G's, T's and C's and we will also put in the tube an enzyme, this is a protein, and it is called a polymerase, and this polymerase is basically going to assemble the A's, T's, G's and C's, and we are going to put one more thing in, which is referred to as a primer, and that is a very small piece of single-stranded DNA, usually about 20 bases or so. It can be longer or shorter, but that is just an example. So that we now have the components to make more DNA. This is what we are going to use to make more. We need our starting material, we need the building blocks, we need something to put the building blocks together, which is the polymerase, and the primer serves as an anchor for that polymerase to hold on to.
Let me stop you for just a moment, Dr. Cotton. With regard to this term "polymerase," is that the first word of PCR, the process itself?
PCR stands for the polymerase chain reaction and basically you are going through a cycle of steps which is why it is referred to as a chain reaction. You are repeating a cycle of steps over and over and over again. And what we will do here is just show you what one or two cycles looks like.
So the first thing we will do is heat this DNA back up to about 95 degrees, enough so the strands will separate.
Yes. And at that will point we will allow the short primers or anchor points to bind and this is--these primers are designed so that the sequence of DNA that they have to hook up to is known and so you have a single-stranded piece in the primer, binding to this now single-stranded starting material, and all the A's and T's and G's and C's are pairing up. And then the polymerase will come along and directed by the bases on your starting material will reassemble a second strand.
Dr. Cotton, if you could, could you briefly describe again what you've already described and perhaps you can point to the various portions of the chart so that each of the jurors may see it.
Okay. We have double-stranded DNA starting material. We are going to heat the two strands, we'll just put a little "x" here on our starting material strands, drop the temperature down, allowing the primer to bind, the two primers to bind, and then allowing the polymerase to make a copy of this starting strand. So the polymerase is basically going along and says I have a t on my starting strand, I will put an a in. I have a C on my starting strand, I will put a g in. I also have a g on my starting strand, I have to put a C in, and so on. So now where we originally had a single strand to start out with, we now have two. Now, I'm--
--I'm sort of going to run out of a length of paper, but the idea is that you then start the process all over again, so you go--I have to stand on this side--so you go, start the cycle again, heat it up. These two double strands will come apart, these two double strands will come apart and now again you let--drop the temperature down and let the primers bind. And I have just made these shorter because there is not enough room. They wouldn't really be shorter, they would be the same length. And then allow the polymerase finish off making the copy for each one of those and so now we have four completed strands where we only started with one. So as you can see, each time you go through the cycle, you are going to double the number of strands that you started out with. If we start out with one, then we would go to two and then we go to four and the next cycle you would go to eight and so on.
All right. Let me--perhaps you can stand on the other side so that I can ask you a question, a few questions about this process. You have used the term "primer." Does it act as something like a primer in priming a pump, for instance?
Well, it is not a bad analogy; it is not great. What it is, is the--the characteristics of the enzyme are such that it needs an end to start from. If you just separated the strands, it wouldn't have a place, it doesn't have an anchor point, so the primer is serving as an anchor point for the enzyme to then connect the next base to. It needs something in the way of the start of a second strand to actually connect, so the primers are in there both to say this is the section we want to copy. That is really what the primer does, is it identifies the section that you want to copy and then it starts as an anchor point for that enzyme to sit down and then move through and copy that piece of DNA.
You have designed your system, that is, you have to know up front the sequence of bases of the piece of DNA that you want to copy. There has to have been enough research done and development done so that--at least in the forensic setting here, you must know ahead of time the sequence of bases that exist in the piece that you want to copy. That has to be done ahead of time. Then you can manufacture in the lab the primers that have the sequence at the beginning and the end and then you are set to go.
As far as these known sequences of where it starts, for instance, and you mentioned you have to know the sequence, have scientists looked at these areas to determine their sequences?
Yes. The development work for the genetic locations that are used for forensic testing with the PCR methodology, these regions have all been sequenced and a lot of other work, besides the sequencing, has been done to study these regions before applying that information to actually designing a forensic test.
Not with this particular polymerase because this polymerase comes--is able to work at a very high temperature, but all our bodies have a similar enzyme so that when a cell replicates and divides to form two new cells, in the process of doing that you are replicating the DNA and the enzymes that perform that replication are also called polymerases.
Would it be correct to say then that this PCR process simply does outside the body what the body does on its own?
All right. Now, your Honor, if we could, I would like to put that drawing pad back where we had it yesterday and then continue on with some additional prepared charts.
Perhaps you could have a seat again on the witness stand, Dr. Cotton, but probably not for too long.
Now, as far as this use of PCR, and I will step over here, Dr. Cotton, as far as this use of PCR in forensics, to your knowledge do we have some charts, prepared charts, that illustrate how you actually use this technique to type samples in forensic case work?
All right. Your Honor, at this time I would ask to be marked as People's next which I believe is--
Your Honor, for the record, this diagram can be labeled or can be described as "PCR analysis," and then showing at the top two large blocks, "step 1, extraction" and "step 2, amplification" and then a smaller block labeled "step 3, "detection."
All right. Dr. Cotton, with regard to this chart that has been placed on the board, have you had an opportunity to look at this before this morning?
And with regard to this chart, does it illustrate various steps in the PCR-based typing process in forensics?
Now, in particular--well, are there a number of steps that occur in this typing process?
In general terms are there fewer steps than in the RFLP typing process that you described yesterday?
Okay. This board itself, and it is labeled at the stop "step 1, extraction." Can you describe what that refers to, and if there is a pointer there that would help you, please feel free to use it.
The top rectangle is simply illustrating that you have some kind of a stain and from that you do a DNA extraction and the DNA is going to end up basically in your test-tube and these tubes are actually very small. And then to that tube you will add, in addition to adding your DNA, you will add what is diagrammed up here as the PCR mix and the PCR mix is the DNA components, the A's, T's, G's and C's, the polymerase and the primers and some other salts or buffers, so all of those things go together in a single tube.
At that point you take that tube and put it in what's called a thermal cycler, it sort of looks like a cash register actually or a small one, but it has a metal block which is illustrated right here, (Indicating), and a computerized programmable method for controlling the temperature in that metal block, so the tube sits down very tightly. The holes in the block are simply the same shape as the tube so that when you put the tube down there, there is close contact between the tube and the walls of the metal block, and the temperature in the tube is then controlled by heating or cooling the metal block and you do this in a cyclical manner. And the cycle goes as we talked about a little while ago. You are heating the block up to 95, so the contents of the tube will be at 95, so you can denature the DNA, separate the strand, and the temperature is then dropped down. And I am not going to be able to remember what the exact temperatures are in the cycle, but the temperature is dropped down somewhere usually around 60 or 64 or 65 degrees, it could even be lower than that--anyway I don't remember right now--to allow the primers to bind and then the temperature is raised up to around seventy degrees to allow the enzyme to complete copying of the strand and then the cycle starts all over again. So you tell the machine in your programming what specific temperatures you want it to cycle through and how many cycles you want it to go through.
In other words, this machine, you can program it or tell it basically how many of these cycles to go through, at what temperatures and so forth?
That's right, and how long each cycle is to be, like you are going to have it at 95 degrees for one minute and then you will drop it down to 55 degrees and stay there for thirty seconds and then you will raise it up to 70 degrees and you will stay there for, you know, fifty seconds or something like that.
This heating and cooling process that you described, was PCR process used before there were machines that did this in a fairly automated fashion?
It was and my understanding was that when PCR was developed that basically the people were doing this by hand, that is, holding the tube in a water bath at a hundred degrees and then taking it out and putting it in another water bath at another temperature. So it would have been very tedious to do that. You would have a lot of researchers standing around taking up a lot of their time holding their tubes in water baths, so anyway, the computer age came and this machine was developed to essentially take care of that for you. So you put your tubes in, you set the machine up and turn it on and it goes through as many cycles as you have programmed it to do and then it holds your tube at a cool temperature until you come and retrieve it.
Just briefly, there is a step 3 listed, that is called "detection." Is it your understanding that we will return to that in some detail in just a few moments?
All right. With regard to this amplification process, and in particular how these strands are copied, would it aid you to use a prepared diagram showing two different double helixes or ladders?
Okay. Then if I could, I would like to return to the drawing pad. And Dr. Cotton, could you use the diagram and let's--let's add a new page if we could that I believe would be People's--
And with regard to this, if you could just illustrate and I would like you to actually write down a series of--just for illustrative purposes or examples--bases and how in this copying process how the primer would then act to add these bases that are floating around in this tube mixture. Do you understand what I'm asking?
You want me to take a double strand, take it apart and show how the second strand is reconstructed?
Okay. So I've made a very short sequence and I see there is a flaw in my thinking here, but we will sort of try to work past that. Double-stranded A's and T's, G's and C's nicely paired up and heated and create the two single strands. We are just unzipping the zipper. And now we want our primer to bind, so here is the flaw in my thinking. I'm going to have to make for a very short primer, because I didn't draw a very long sequence, so let's say we have put in a primer here, (Indicating), to have two T's and we put in a primer to fit the other side, which is going to be a t and a G. And you can see that this primer won't attach to this side and this primer won't attach to this side and the primers won't attach to each other, so you have to take these things into consideration and then you simply have your enzymes come along and the enzyme will come along and create this new strand. It will use this primer as its anchor point and now add--and remember we have got, you know, just individual A's, T's, G's and C's floating around here available to be put into the new DNA strands, so the primer will come along and correctly make a brand new strand on both sides.
I said primer but I meant polymerase. The polymerase will come along, hook onto this primer and correctly make--finish off here the new strand, (Indicating).
KEY QUOTESo what you have illustrated is basically how this process works with the use of the primers and how from one strand you create two strands?
And then that process just continues through the cycles a number of times over and over that you described a few moments ago?
Just a couple more questions if I could, Dr. Cotton. With regard to this primer, it latches onto the other side of the DNA strand; is that right?
It will bind--there is no other side. This is a single strand now and this is a single strand at the point that you've heated it, so the primer is the first starting point to creating another side.
And it is from the primer on up or down the ladder, as the case may be, that it then starts this process of adding these bases one at a time?
All right. Very good. With regard to--and now just referring very previously to the large chart, which I believe is People's exhibit 248, as far as this--and you have described the second step on this chart, "amplification"; is that right?
With regard to the third step, is that the final basic step in this process of PCR typing?
That's right. You are going to--now you have made lots of copies of your DNA and you want to look at it for purposes of forensic use. You will be copying sections of DNA that have some difference from person-to-person and now you want to figure out, well, what does that difference look at for the sample I just copied.
In other words, you actually determine the types in a particular sample so you can determine what persons can be excluded or included as possible donors?
With regard to this detection possess, is it the same or different from what you are looking for using the RFLP method?
Depending on the genetic location that you have amplified, there is--one of the detection methods is similar to the RFLP. That is, you are simply looking at how long a piece of DNA is you amplified. You know you started at one end, but there is some variation in the length, and you may need to explain that a little bit more. But the other--the other type of test that is available does not have anything to do with length really, but has to do with a sequence difference, so that at a particular genetic location I might have one sequence, Mr. Clarke might have another, Mr. Goldman, might have another, and so could you look at the sequence differences.
Thank you. Your Honor, for the record, People's exhibit 250 could be described as labeled "DNA" with what appear to be two depictions of a ladder side-by-side, DNA ladder.
Dr. Cotton, with regard to this exhibit, first of all, have you had an opportunity to see it before?
Would this exhibit help you describe the differences between these two methods of typing samples that you just made a brief reference to a moment ago?
Yes. It is--it is the good example for--and it--it is a point of discussion for what is a sequence difference.
Yes. What we have here is the same diagram basically that we were using yesterday do show what is a DNA molecule and here we just have two of them that are side-by-side. And if you just go, let's say, from the top to the bottom and on one you will see we have an a t pair at the top and the other there is an a t pair at the top and we come down to a GC pair and the second one also has a GC pair, but here in the middle the DNA molecule on the left has an at pair. The DNA molecule on the right as a GC pair, so-- and then if you go on down the remainder of the three base pairs here, they are alike and a GC, an at and another GC, so the only difference we have here is the fact that the third base pair down on one diagram is an at and on the other is a GC. This is a DNA sequence difference. This sequence difference can be detected because you can amplify both of these pieces of DNA and then use a detection method to allow you to--you match either these pieces or those pieces and you can figure out that you have two varieties basically in your sample. You have one molecule that has an at pair and another one that has a GC pair, and so in the PCR diagram that we were looking at a little while ago, when it got to the bottom and it says "detection," this--to amplify two pieces of DNA and then detect that they had a sequence difference is one type of detection that is used for forensic analysis.
What is the other type of detection method that the board just named, very briefly?
The other type is looking at a length difference, which is very similar to what you are doing in RFLP. You are also looking at a length difference. But maybe we should go back to the piece of paper just very briefly.
Your Honor, for the record, the witness is labeling this as "PCR length difference."
What I'm going to do is go back very briefly to the same analogy or the same example that I used yesterday in terms of a repeat, so let's say we have the same C, A, t repeat and again I'm just using that because it is a very easy example, and I have two different DNA pieces here, and they--one has two repeats and the other has four. And again, if I can somehow identify the end of each of these pieces, I could look and see how long they were. For the RFLP test we defined the ends by having an enzyme come along and literally cut these sections out. You can use the PCR test to also look at the lengths and you can do that by designing your primers so they will come along and bind just outside the repeat. Now, we could go through and redraw all the amplifications, but if you think about if, we look at just one of the strands here, if our primers come along and copy this section and copy this section, (Indicating), ultimately as you go through the PCR you will end up with a piece--many pieces that are this long, (Indicating). If you do the same thing with the other piece, with your primer sitting just outside the repeats, the pieces that are eventually going to become your product or your many pieces of DNA that you have now copied will include the full length of the primers and the four repeats that I have diagrammed, whereas over here we only had two. Now, instead of having a sequence difference like the board that we just talked about, now you have PCR product, but the difference is a length difference and you can analyze that length difference by separating the DNA strands to a gel. It doesn't always have to be exactly the same kind of gel that we talked about yesterday, but the principle is exactly the same and that is the DNA strands will move through the gel and separate out according to how long they are. And that is the other commonly used method of detection for looking at your PCR product, that is, looking at some kind of genetic difference in the DNA that you have now amplified.
As far as the sequence differences, is my sequence difference at a particular set of markers different than yours?
It may be and it may be the same. Since we haven't tested both of us, I couldn't answer that question.
The same thing. The variation in the population for these PCR markers is not extensive, so many of us may share some things and then some of us would be different, so whether or not you and I are different, I couldn't tell.
Do you look at multiple genetic markers, more than one genetic marker, to get these differences if they exist?
You look at many genetic markers as you have the ability to look at, and no matter what kind of system you are using, be it PCR or RFLP, the more markers you look at, the more genetic locations you analyze, the more information you have. That is a generalization that will always be true for forensic testing. The more markers you look at, the more information you have.
And is that your goal as a forensic scientist, to look for as much information as possible?
Is also labeled "PCR analysis" but it has two small blocks at the top, "step 1, step 2" and then a large block, "step 3," for the record.
Now, Dr. Cotton, with regard to this chart, People's exhibit 252, would this help you in describing how these actual differences, whether they are sequence differences or length differences, are determined by you as a forensic scientist?
All right. Then if you would, with this chart, then describe for the jury these differences in typing and how they work.
Okay. So step 1, we extracted the DNA. Step two, we amplified it, we used the thermal cycler, and like you saw on the previous diagram. And now based on whatever genetic location we amplified, we are going to use one of these two types of detection, and the first type that is illustrated, which is on the left, is called a dot-blot or sequence polymorphism. That is sequence polymorphism just meaning sequence difference. That is exactly what we looked at on the two DNA molecules just a few minutes ago. So the set-up is you have a nylon strip which has DNA bound to it in specific locations and that is illustrated right here, (Indicating). There is a plastic tray and the tray has like little wells in it, sort of, and the strips sit in the tray. Solution is added to those strips and your amplified DNA is added to those strips and the DNA that is spotted onto the strip will bind to whatever part of your amplified DNA that it matches up. And where the DNA binds to the strip, the strip is then processed through a series of reactions to give you a blue dot on the strip where your sample DNA bound and these blue dots are then interpreted to say you have--and this is a term we haven't used--when I talked about genetic differences, if you go back to just mother and father, if you have two differences. Those are referred to as an allele. An allele is simply a form of a gene so that, for example, the two sequence DNA differences that we showed just a few minutes ago, those could be called two alleles, or the term Mr. Clarke was using would be two types. So from the series of blue dots on the strip, you will read off the types or the alleles for that DNA sample that you just analyzed. Then you simply write those down and record those. The other method, which is diagrammed on the right--
Your Honor, I would ask that be marked as People's next in order what can be described as a tray containing various slots in it.
Dr. Cotton, showing you what will be marked People's--I'm sorry, was that 253, your Honor?
The item is the typing tray. It is the tray that is illustrated in the top two pictures here. It is plastic. This tray has strips laid in the wells. The strips have not been processed, so there is no blue dots on them. And the tray has a clear plastic lid which I taped down so that I wouldn't lose it, and this is the typing tray that is used in the laboratory and examples of the strips that are used in the laboratory to go through this series of reactions of adding your amplified DNA and developing the blue dots on the strip and from those blue dots then you would read your types from those samples.
Where did that tray actually come from, as well as--I'm sorry. Did you say there were strips inside the tray right now?
Would that be a tray and strips then that you would have used in case work if it hadn't been brought to Court today?
With regard to those strips--and they are currently laying in the bottom of the tray; is that right?
The strips go in first and a solution that has some salt in it goes in and your amplified DNA goes in and that is incubated in a water bath so the water bath is controlling the temperature. The water isn't coming over the tray, long enough for your amplified DNA to bind to the DNA dots that are on the nylon. Then a series of reagents or components are added which allow--well, first the extra DNA is washed off and then a series of reagents are added to allow the development on the strip of the blue dot from your DNA bound, so you go through adding your amplified DNA, pouring off any that didn't bind, adding in a series of reagents that you need to develop the blue dots, pouring that off and then allowing them to develop.
And then finally do you see the appearance of these blue dots? As is illustrated, they're the bottom left-hand side of People's exhibit 252, the large diagram?
All right. Your Honor, at this point I would ask that this exhibit be allowed to be distributed to the jury.
Dr. Cotton, if you could, could you then turn to the right-hand side of People's exhibit 252. And does that describe a different detection method or typing method when you are looking at these length differences?
First of all, at the top it seems to have some letters and numbers. What are those?
The "AMP-FLP" is sort of a shorthand version of saying amplified length polymorphism, "amp" referring to amplified, "f" is fragment, "l" is length, "p" is polymorphism. D1s80 is the--is the nomenclature for the genetic location that is currently used in forensic labs that has a length polymorphism.
We will return to that later, but is d1s80 simply a way of describing a particular genetic marker?
Okay. So in this case you have amplified DNA. The pieces that you amplified may have different lengths and so you are using a gel, which is our green gel here with the red DNA on it, and the DNA is loaded into the gel. It is a slightly different type of gel than the one I described yesterday, but the principle of how the gel works is exactly the same, and the gel is subjected to electrophoresis and the current moves the DNA through the gel. It moves through based on its size. And in this case you don't have to use p-32, you don't have to do anything fancy after you have run the gel. You can simply use a stain and visualize where the DNA fragments are, so at the end you have a gel that is stained and you can actually see the DNA fragments directly. And then you can photograph that gel and you can compare the positions of DNA bands in the various samples that you have now analyzed.
And that is comparison of the bands like that same process of comparing bands using the RFLP method?
Now, with regard to this length difference and this typing method described on the right, is that again simply one of the means of typing a particular genetic marker following the use of this PCR copying possess?
As well as this dot-blot method used to look at differences between people, whether it is based on a sequence difference?
That's right. With--with the RFLP everything is--you are looking at is length. When you use PCR, you can use a test that looks at sequence differences that is illustrated on the left or you can use a test that looks at length differences and that is illustrated on the right.
One more question, if I could, and particularly while you are up, Dr. Cotton, I would like to take you back to the current drawing, which I believe is People's exhibit 251 labeled "PCR length difference." With regard to that particular drawing, you described the use of these enzymes to cut the DNA at certain locations?
I simply made reference to that in that for PCR--scratch that. For RFLP, to define the ends, that is to define the length, you use an enzyme to cut that length out. For PCR, to define the length,--to define the ends, you are using the primers. That defines the end and then you are copying however many repeats happen to be in between. And whether you have a few repeats or a lot of repeats, that determines the length.
The primers don't really know anything, but they are simply a piece of DNA that is single-stranded and they are going in and--and binding with the piece of DNA in the reaction tube that matches them. That is why my short primer example wasn't very good, because to be very specific, the primers need to be longer and they are usually around 20 or possibly more bases.
But for purposes of showing how this process works, that is why you used shorter lengths or shorter sequences?
Your Honor, it was my intent to clear the charts and enter a slightly different area.
Dr. Cotton, as far as the use of PCR in your laboratory, was there a time when you actually began using this technique in your actual case work?
We started doing PCR in case work in about--let me think about this a minute. I believe it was about June of 1992.
And how do you decide--if a sample or a case comes into your laboratory, how do you decide whether to use the RFLP technique or to use the PCR copying process followed by typing genetic markers that PCR looks at?
For our particular laboratory, that may be related to a lot of different things. Umm, for example, it may be that another lab has--had already done RFLP, but doesn't have the capability of doing PCR, so they might specifically be asking us to do PCR. More typically, we would get a piece of evidence in and we would have to make an assessment. Is this piece of evidence--does it have enough DNA and is the DNA in sufficiently good condition to do RFLP? And if so, then you would proceed with RFLP. If the DNA or the piece of evidence contains only a very small amount of DNA or the DNA is very degraded, then you would choose to go forward with PCR.
That is what I was just going to ask next. Do they each have their own advantages, these two different approaches?
For RFLP you need a larger amount of DNA and it needs to be in very good condition.
Because the test--if you remember yesterday I was talking about lengths of DNA that were thousands of base pairs long. You are looking at--in our lab we are looking at a piece of DNA that is anywhere from about 1500 up to 12,000 base pairs, so your DNA has to be in good condition to even--it has to be much bigger than that as starting material or your test won't work, so it has to be in good shape and you have to have a fair amount of it. The RFLP test has the big advantage in that it is very discriminating from one person to the next. There is so much variation in the population that each time you look at another genetic locus or genetic location with RFLP, you are getting a lot of information. That is, you can discriminate one person from the next with a high level of discrimination. On the other hand, the PCR, the tests that are currently available doesn't have that level of discrimination. It is a much lower level of discrimination, sort of in the range or maybe somewhat better than a whole series of serological markers. However, the PCR can use DNA when you have a very small amount or it is in very poor condition or both.
So in other words, in terms of the ability to tell many of us apart as human beings, in other words, a powerful test as far as this ability to again tell large populations apart, is the RFLP procedure then the more appropriate to be able to determine that type of information?
As far as the PCR process itself--and let's talk about both techniques in terms of how long they take to obtain results. Are there any differences between the two?
If you brought a sample into the lab and you didn't have--say you didn't have anything else to do, every other case that you were working on was all finished and you simply went through the PCR process, you could have results easily by the end of the week, so it would be very fast. And that is assuming you have, you know, a moderate number of samples, say three or four of something. Easily you would be done in a few days. The same test with RFLP could easily take you three months.
The length of the time it takes to do the RFLP test is pretty standard from the time you extract the DNA until you load it on the gel, but the exposure of the x-ray film to that nylon membrane, if you have a lot of DNA, it can be short, as short as a day, but if you have little DNA, it can be as long as two weeks. So it could be that for the RFLP test you took two weeks to look at one genetic location, then you took another two weeks to look at the second and another two weeks to look at the third and you can see that rapidly eats up a lot of time.
So it is that exposure of creating this x-ray film that takes a great deal of time; is that right?
Are there differences in the time it may take to create this x-ray from a particular sample?
Yes, and the differences are basically related to how much DNA you have in your sample. If you think about a large amount of DNA and a small amount, on your nylon membrane, you add your p-32 DNA probe. If you have a lot of DNA there you can bind a lot of probe. If you only have a little DNA there, you can only bind a small amount of probe. It is the emissions, the radioactive emissions from that probe that expose the x-ray film, so if you have a lot of probe there you will get an exposure on your x-ray film quickly. If you only have a little bit of probe bound because you only had a little bit of DNA to start with, it takes a long time to get enough exposure to that x-ray film to actually see something.
And again, if I can show you what was marked yesterday as People's exhibit 246, is that one of those x-ray films or autorads that can require this fairly lengthy period of time to develop?
Now, you have described a little bit about the use of genetic markers and you actually identified one of the particular markers and I believe it was d1s80; is that right?
Is that just simply science's way of designating markers to tell one from another?
Is there any particular reason there is numbers and letters in it instead of just letters like PGM?
The DNA locations that do not code for--do not have information for a gene that we know of, are given letter designations. The "d" stands for DNA. The "1" is because that particular location is on chromosome 1 and the "s80"--I actually can't remember what that refers to--but that also has a specific reference. So--and they are called anonymous DNA pieces, that is, they are not a gene that we know about and they are just given these "D" designations, it is an international nomenclature, so that if I read a journal article and it has one of these probes, it might say D5S20 and that would tell me that that DNA location was on chromosome 5 and if I could remember what the S20 meant, it would tell me more than that.
As far as these genes that you say have these descriptions like starting with a "d" and then a number of the chromosome and so on, you said that something about them was not known. What is that?
Most of the--in fact, as far as I know, all of these genetic locations that contain these repeats, these are not genes in the traditional sense. Biologically speaking, in the traditional sense, a gene is a piece of DNA that contains information that creates a protein that then can go out into the cell and has a function. We know that these repeated sequences are not genes in that sense. Scientists don't actually know what the function of these repeats are. That doesn't mean they don't have one; it just means we don't definitely know what it is.
As far as forensic science, and in this process, do you have a process of deciding or selecting which genetic markers to look at for purposes of identifying people?
Yes. For all of the markers that are currently used, there has been a substantial development period. That is, of the markers that are available, people have gone in and done the kind of research to say how much variation is there in the population for this marker, how easy is this marker to use, how much development do we have to go through so that many laboratories could have access to this particular marker. So this kind of work is done in a development sort of stage and then a marker is collected because of its utility, its ease of use, its discrimination power and so on, to be then used in--in a wider setting with--where many labs have access to or they choose to use a particular marker.
As a forensic scientist would you, for instance, normally be interested in looking at the cystic fibrosis gene for purposes of your work?
Although there is a lot of variation in the cystic fibrosis gene, that variation isn't widespread enough in the population so that each time you used it you would expect to get very much information. It wouldn't be sufficiently informative for forensic science purposes to use it.
So is it correct then that the genetic markers you are most interested in are those that show us differences between people and can be easily used and are able to be typed, for instance, in older samples?
Now, let's go back to PCR itself. Does it have uses outside forensic science or is it just limited to work in an area such as yours like human identification?
PCR is sort of the next thing that came along after this southern blot that is used everywhere. The use of PCR in forensic science would be a very tiny fraction of its total utility to biological research.
PCR is used in the same kind--in genetic analysis, in gene mapping and it is used in many basic research settings simply because it allows you to get a whole lot of DNA from a very small amount of starting material, and whenever you can Get--scientifically, whenever you can get a lot of something, it gives you the ability to study it, so if you want to look at a particular section of DNA, then to get a lot of it makes you work a lot easier.
You raised the term or you used the term "genetic diagnosis." Is that determining whether an individual, for instance, either has a genetic disease or is I think you used the term a carrier?
That is if a parent has a genetic disorder, then the parent can transmit that genetic disorder to his or her children and this is something that many parents want to be aware of before they start having children, and you know, so you might say shall I have--a family might decide will we have children or will we adopt children, because one or the other of the parent could be the carrier of a genetic problem.
Is PCR used, for example, to counsel parents, as you have just described, about the likelihood of a child of theirs having a genetic disease?
I'm sure it is. I'm not a good enough plant biologist to know any specific applications, but I have no doubt that it is being used.
In terms of understanding human diseases and human genetics, one of the primary things that scientists need to do or want to understand is how are genes located relative to each other, what gene is on what chromosome, what genes reside close to. And so in the process of figuring this--answering these questions for a specific genes, PCR will be a very helpful technique.
Is PCR used, for instance, in the identification of the remains of American war dead?
All right. Ladies and gentlemen, we are going to use this point to take a brief Court reporter recess for the morning. Please remember all my admonitions to you. Don't discuss the case among yourselves, form any opinions about the case, conduct any deliberations until the matter has been submitted to you, or allow anybody to communicate with you. And we will resume at ten minutes until 11:00. All right. Dr. Cotton, you can step down.
PCR is a way of saying I want to replicate a small section of DNA and it allows you to replicate a small section of DNA in a way that if you have a very small amount of starting material, you can go through sufficient number of replications so that by the end of the process you will have enough of this small section to then do some analysis on.
The more markers you look at, the more information you have. That is a generalization that will always be true for forensic testing.
Absolutely. Then that would be the test of choice... For RFLP you need a larger amount of DNA and it needs to be in very good condition... the PCR, the tests that are currently available doesn't have that level of discrimination. It is a much lower level of discrimination.
I said primer but I meant polymerase. The polymerase will come along, hook onto this primer and correctly make--finish off here the new strand.