We are --we are a private laboratory and we do paternity testing and testing on biological evidence.
I have a Bachelors Degree of Science with a major in Biology. I have a Master's Degree of Science which major in biology and both of those are from Southern Methodist University in Dallas, Texas. I have a Ph.D. in molecular biology and biochemistry from the University of California at Irvine.
It's basically a set of techniques that allow you to study the molecular interactions that occur in cells that allow cells to function.
Okay. And in your studies to obtain your Ph.D. in these fields, did you deal at all with DNA?
DNA is packaged into chromosomes and my work at Irvine was involved in looking at the proteins that helped to package the DNA up to form the chromosomes.
I went to the University of Iowa and did a post -- Post Doctoral Fellow which is going to someone who has an established laboratory and working in their laboratory and I worked there in the Department of Biochemistry for three years.
I went to the National Institute of Health in Bethesda, Maryland and I worked there for about four and a half years.
And what did you do while you were -- Well, first maybe you could tell us what the National Institute of Health does?
It is a government funded research institute which is really composed of many smaller institutes. There's probably, I don't really remember, but maybe 3 to 5,000 scientists. It sort of looks like a good size college campus. And it's -- all the people there are involved in government funded research which is mostly health related.
I was involved with a unit that was looking at the genetics of alcoholism and we were mapping some of the genes that are not necessarily involved in alcoholism, but involved in how alcohol is metabolized when it's -- when you've had some.
If a scientist or a group of scientists does some research in the laboratory and they want to publish that, you write up what you did, the results you got, how you got them and send that off to a scientific journal.
The journal then sends that out to two or three, usually other scientists, who are also knowledgeable in the same area that the paper is written in.
And those scientists read the paper, make comments and then send it back to the journal, and the journal is basically asking, should the paper be accepted; should the paper be rejected, or should the paper have more work done to it and then re-submitted.
The articles that you've written and articles that you review, are they all involved with the DNA?
The ones I would be peer reviewing, currently would be involved with DNA as it's applied to DNA testing, as it's applied to evidence.
Couple -- couple years later, I became the Deputy Director, that is the Assistant to the Laboratory Director; and some years after that I became the Laboratory Director.
And can you tell me, generally speaking, what your duties are as a Laboratory Director at Cellmark?
I have overall scientific responsibility for the work that's done at Cellmark. I supervise both paternity and forensic, both testing of evidence work. There are two supervisors who work underneath me to help me do that.
And I oversee every research and validation work that's done at Cellmark and I testify when necessary.
Cellmark handles about a thousand or so paternity cases per year and about 500 forensic cases per year.
(BY MR. LAMBERT) And are the cases that are handled by Cellmark, are they done pursuant to any -- especially the forensic cases, let's take, are they done pursuant to any national guidelines of any sort?
The guidelines are referred to as the TWGDAM guidelines. It's T-W -- all capitals T-W-G-D-A-M, which stands for the Technical Working Group on DNA Analysis Methods. It's a group that was formed by the FBI, specifically to write guidelines for testing evidence samples using DNA analysis.
Proficiency testing is having some samples come into the laboratory where someone else knows what the answer should be. So the laboratory is receiving the samples. We generally know that these are proficiency test samples, but we don't know what the answer is, so we do the analysis and report that analysis back to the submitting agency.
Many other labs will do the same proficiency test and then all these results are compared.
In 1988, we did a large proficiency test sample set and got one incorrect match. And the same thing occurred in 1989. We also did a similar large set and got one incorrect match.
KEY QUOTEWe made some changes in our protocols to address the problems that we thought caused or the -- to address the proto -- protocol steps that we thought had caused these problems.
In addition to the TWGDAM guidelines concerning proficiency tests, are there other guidelines that laboratories must follow if they choose to follow the TWGDAM guidelines?
No. Analysis of evidence -- the TWGDAM guidelines are pretty much the nationally accepted set of guidelines.
Now, could I ask, if you're a member of any -- if you're a member of any professional organizations or societies?
I'm a member of the American Association of Blood Banks, the connection there is that they have a paternity testing set of guidelines and we do paternity testing. I'm a member of the American Association of Human Genetics and the American Academy of Forensic Science.
Basically, it's a large group of scientists who have interests, scientific interests in a broad area of human genetics.
The American Society of Forensic -- American Academy of Forensic Science, what does -- is that involved with?
It's also a group of scientists that have scientific interest in the application of scientific techniques to the analysis of evidence.
Basically, the application of science to answering questions regarding some type of forensic evidence.
Now, as part of your job as Cellmark Lab Director, do you testify in court as part of that job?
I don't keep an exact record, but I testify about 12 times a year. I probably testified over 100 times.
And in all of those times that you've testified, have you been qualified as an expert witness?
Now, we've talked already a little bit this afternoon about DNA. Perhaps you could help us out by telling us exactly what DNA is?
DNA is found in the nucleus of all of our cells. It's found in basically all organisms, and the DNA molecule contains all of the information that allows our body to function.
One half of that information is inherited from your mother, the other half of that information is inherited from your father. And the combination of that information carries the code that tells your body how to develop and what it will look like and basically most everything, unless you want to talk about the behavior; and that's sort of up for grabs.
It was first discovered in the early, late 1800s or early 1900s. And I don't really know the exact date. The structure, as we know it today, the chemical structure was discovered in 1953.
Since the discovery of the structure of DNA, has that been the subject of scientific investigation?
Since 1953, yes, there have been many advances. We know a huge amount now that we didn't know -- well, I didn't know anything in 1953 about DNA, that is.
KEY QUOTEOn the left, we'll start on the left with the cell. This one sort of looks like an egg, but that's fine.
All cells, except red blood cells, have a nucleus. Red blood cells lose their nucleus in maturing to be a functional red blood cell. And when you get DNA from the blood, you're getting it from another cell type which does have a nucleus.
The DNA is found in the nucleus, packaged up into 46 chromosomes. 23 of those, you -- each of us inherit from our mother and 23 of those each of us inherit from our father.
So the second panel just shows a lot of chromosomes sitting in this nucleus that's been enlarged. And the next panel over -- shows what the structure, sort of a cartoon structure of what a chromosome would look like if you blew a single one up in a microscope, and were able to see that. And you can certainly do that. In fact, many people are very expert at that.
In that chromosome, the DNA molecule wound up. And so in this chart, the DNA is depicted here as blue and it's shown -- sort of shown to look like a slinky. That's maybe -- that's another sort of cartoon version. But there's some accuracy to that. If you further unwind that slinky, what you wind up with is have -- have a very long thread of DNA.
That's what's shown in that final panel that the DNA has a helical structure as if you took a ladder that was not made of metal and just twisted it around, or if you took a ribbon and made some twists in that ribbon, you would end up which double helical structure.
Perhaps we could have the next chart then, please. I think this one is going to be the new number. So it would be?
This is just a really short section of a DNA molecule diagrammatically, but it's pretty accurate, and there's a couple of things that are relevant in this diagram.
One is that the DNA strand is made up of only four components and they are Adenine, Thymine, Guanine and Cytosine and every scientist in the entire world, refers to these as ATG and C, by the first letter of their chemical name.
And these four components, if you just looked along the whole strand, the order of these components actually makes a code, and that's what carries the information. So instead of having a 26 letter alphabet, DNA has a four letter alphabet.
The components are in the molecule in pairs. So at the very top you see a green "A" and a gold "T." And the next one down you see red "C" and a blue "G." Those -- And then you see another AT.
A and T are always paired together, G and C are always paired together. And that feature of the molecule allows you to do something in the lab that's necessary for DNA testing, which is you can pull the two strands of this helix apart, as you would unzip a zipper and put them back together. But they only go back together when the AT and GC pairs are all properly lined up.
So it's like if you think of a zipper that has different shape teeth that sort of fit together like a lock and key, with an A and a T forming a lock and key, and a G and a C. But the shapes are slightly different for the AT pair, than they are for the GC pair.
When you pull these strands apart and you can put them back together and that feature of DNA allows DNA testing to be possible.
There's only one other feature that's really important in terms of thinking about DNA testing as it's applied to forensic or paternity, and that is you can think about -- you could measure -- there are various scientific ways to measure the lengths of DNA. But when people do this, they don't talk about a piece of DNA being so many millimeters or so many inches long. They talk about how many base pairs it is.
An AT pair or a GC pair is called a base pair. It's almost impossible to talk about DNA without interjecting that scientific terminology.
So the piece that we're looking at on the chart has six base pairs altogether and you can talk about length -- the length of a piece of DNA and you always talk about it in terms of how many base pairs long it is.
In humans, there are about 6 billion base pairs of information that makeup the whole human DNA, that exists in all of the chromosomes.
There are actually many more similarities than there are differences. At least 99 percent of DNA in humans is the same. We all have five fingers on our hand. We all have the same body parts. Our bodies all function similarly. So the great majority of DNA in humans is going to be the same between myself and everyone else in this courtroom.
However, a small fraction of the DNA is different and that's intuitively obvious because you can look around and recognize pretty quickly that people don't look alike unless they're related. And even if they're related, they don't look identical.
With the exception of identical twins. Identical twins look identical because, in fact, they have identical DNA.
KEY QUOTEAnd this portion of the DNA that's different from person to person, is there a way to determine the differences by looking at that DNA?
Well, let's take a look at the next two charts and maybe that will help you. Might bring them both back, Steve.
Yes. There's two common ways, two common types of differences in DNA that are used in both paternity testing and forensic testing. And this first board shows one of them. And this is the idea that a particular section of DNA is different. People will be different lengths.
Can I --
This is the same little sequence that we saw on the diagram that you had up just a few minutes ago. And it's got four base pairs here, a GC, an AT another GC, another AT. And what the diagram shows is that you've got four of those right here. You've got four of those sections all stuck together. So this four base pair section is repeated four times.
Sort of like box cars on a train. This train has four box cars. This chromosome over here -- let's say that, suppose this is my DNA and this is one of my chromosomes. And I got this one. I got this chromosome from my mother. This is the corresponding chromosome, let's say, that I got from my father. But here, there are only two sections, two box cars stuck together of this four base pair section. Two pieces stuck end to end.
Now, let's go on and look at another person for that same chromosome pair, for that same genetic location. Someone else might have their sections repeated end to end. And say they got that chromosome from their mother and on the other chromosome they inherited from their father. There were five of that four base pair section stuck end to end.
So here I've got altogether, eight base pairs. Here I've got 16. Here I've got 12, and here I've got 20.
If I had a way to go in to the DNA and cut these sections out that had these repeats, I could see that my DNA was different from this other person.
That kind of difference in DNA is one of the differences that -- current methods for DNA testing looks at.
And this, the difference that you're describing is the difference in length of these repeating sequences?
That's right. So this is a length difference or a length. The scientific term is a length polymorphism. Polymorphism means several forms. So this is an example of a length difference from one person to the next.
So, would you say that the person one and person two are polymorphic in this example? This one up here?
Could we look at the next one, please?
Did -- this one does have an existing number. It's number 276.
On this one we've only shown what would be only one person. You get two copies of every chromosome, so one copy from mother, one from father. But this serves to show the idea.
Here, if we look along one side, we have a C, a T, a G and an A. If we go to the other DNA strand, we have a C, just like over here, but here where we had a T on the first one, we have a C on the second one.
So we have a difference in the sequence of the basis or the base pairs as you go down the molecule at this particular location.
And someone else might have the same C or the same T, or they might have a G there. And I'm just reading down one side to be consistent. I'm just picking this side, the left side, to my left side to read down.
So this is an example of a sequence difference. Where at a particular location, the sequence of the basis isn't necessarily the same in every person. And that is the other kind of difference that DNA testing in paternity and forensic uses -- makes or uses to distinguish one person from the next.
So Dr. Cotton, taking these two DNA differences that you pointed out to us into account, have tests been developed to determine whether a person's DNA is different from another person's DNA by looking at these kind of differences?
Okay. Why don't we take them one at a time. What's the RFLP test? Maybe you could first tell us what RFLP means.
RFLP stands for Restriction Fragment Length Polymorphism. And the two operative words here are the last two. It is a -- the test looks at length differences.
And could you just generally describe how this RFLP, and if you don't mind, I'll use the shortened version of that how the RFLP test works?
For either kind of test, it doesn't matter if it's RFLP or the other type, PCR. The first thing that happens is that you have a piece of evidence that has some biological tissue or fluid on it that has cells.
The first thing that you're going to do is purify the DNA away from both the piece of evidence itself, like a piece of cotton or a stain on a clothing or whatever it happens to be.
And you also want to purify the DNA away from the other parts of the cells that you're not interested in looking at. You're really only interested in looking at the DNA.
After that, you go through a procedure that allows you to cut out these sections that we know vary in length from one person to the next.
Can we have the next chart please, Steve?
And I think this is going to need a new number as well.
No. I'm told it has a number. What this is a portion of existing 274. It's the last portion of existing 274.
And this chart diagram of Autorad, perhaps you can explain to us what this chart depicts and also tell us what Autorad is?
In this -- in doing this RFLP procedure you're going to purify the DNA, then you're going to go through this second step. It allows you to cut out these fragments that are different in length and then you're going to go to another step that allows you to separate the fragment, based on how long they are.
And then you go through yet one more step to be able to see your results.
The final results are visualized on a piece of X-ray film. You haven't done an X-ray, keep in mind, but it is that same type of film that's used and you see dark lines on this X-ray film.
And the chart that's on the easel is a very simplified diagram of what's shown on this X-ray film, which is called an Autorad.
And can you tell us, using this as an example, what an Autorad can show you about these length differences?
What I want to do, to orient you, there's a, what's called a top and a bottom to this.
There's a starting point for your DNA, and there's going to -- just give us some starting point across the top here, which is actually a place where your sample goes in and the DNA in this procedure moves this way. This is the bottom. This is the top. These fragments at the bottom or anything that you see at the bottom, is shorter. And anything that you see at the top is longer.
For each sample, generally you'll see two dark lines that are called bands, B-A-N-D-S. It's just like -- could be "mark" -- I don't know where the term "band" came from, but it's been around a long time.
The piece of DNA that's resulted in this band is physically shorter in length and different from this piece.
So let's suppose that I have three samples here; one, two, and three. And these are the appearance of the bands that I get from these three samples.
When this result is completed, you can look at these. Sample one and sample two have appearances that look the same, that is, the bands are in the same relative position.
So it's possible that sample one and sample two came from the same person. Sample three has two bands also. Again, each time, you know, one from mother; one from father. Sample three has two bands also. They are in different positions than the bands in sample one and sample two.
So the person with whom the DNA came in sample three cannot be the same person as one and two.
What you're looking at here is the amount of information you would get, looking at one genetic location and that's the starting point. But generally, you would go on and look at two, three, four, five, and possibly more, each providing this kind of information. So you would end up with a group of data, a set of data from several genetic locations. And each time you're asking the same question: Can -- are one and two the same or are they different?
We already know this is different. So that answer's one question right there.
Two and three --
(BY MR. LAMBERT) Now, when you actually use this system in case work, do you make the comparison just visually?
No. In case work, you -- well, you do make the comparison visually when you see it the first time. You have to look at it. And it's relatively easy to make this comparison visually. You don't need any further assistance.
After you've made the comparison visually, there is a computer assisted imaging device that allows you to make some more precise measurements about the proximate size of the length of DNA that's created each of these bands, and then you compare those in a numerical fashion and come to a final conclusion.
This RFLP method that you just described for us, is that one of the tests that you use at Cellmark?
Yes. It's used in -- it's used in a lot of research applications. It's used in some medical diagnostic applications. This very same test is used to monitor bone marrow transplants. So it's not just a forensic application. There are a lot of other applications for this procedure.
Now, this chart, which is teetering up there, is this chart designed to explain the PCR test; is that right, Dr. Cotton?
The big difference in a PCR test is that you don't have as much DNA to start out with. And the only way you can analyze the small amount of DNA that you have, is to make some more of it. Although you're not making more of everything, you're just making more of the particular section that you're interested in looking at.
So if you look at the top panel here, it shows, like, a white cloth with a small stain on it.
And then the blow-up there in pink is trying to illustrate that you're getting DNA from that stain. You're purifying, the same methods that you used to purify the DNA to RFLP are used for PCR and some additional different ones as well.
If you look at the pink circle there with the DNA in it, you'll see that most of it is very dark gray. But there's a short section that's blue.
And let's say that this is the section that we want, that we're interested in looking at. There's something about this blue section that's going to show differences from one person to the next, and we don't have enough of it to look at. But when we'd like to make more and that's what this procedure allows you to do.
And that is, you add the DNA into a tube and you add an additional component that allows you to copy that short blue section.
So step two just shows that you've taken all these components and what you're doing is you're putting them in a machine that cycles through a set of hot and cold -- It's not real cold, but warmer and cooler temperatures that allow this chemical reaction to go forward and make many copies.
And so on the -- on the lower panel here, in the test tube, now the pink circle is trying to illustrate that now I have a whole lot of that blue section that I wanted to make a copy of. And now, because I've been able to make so many copies, it's like xeroxing.
If you want to have 20 copies of one page, you just go in and push "20" on your machine. Well, it's -- that's where the analogy ends. But it's like you can make many, many copies of one section, one page of the DNA, if you want to sort of say it that way.
And now you have enough to actually do an analysis and then the small little panel at bottom here, where it says step three, that analysis might look at a sequence polymorphism, a sequence difference. That's one kind of analysis that is commonly done.
And the other kind of analysis that's commonly done where it says: AMP, FLP is actually -- it just looks at a length difference in the same manner that the RFLP test looks at a length difference, but you just -- it happens that you're looking at shorter smaller pieces, but the idea is the same.
So taking the later point first in the PCR context, is there a particular test that's commonly used to a look at fragment length differences?
It's called D1S80. That's actually the name of the genetic location that the test looks at.
(BY MR. LAMBERT) What I'd like you to do now, Dr. Cotton, is explain another kind of test that is used in connection with the PCR process, kinds of tests that look at sequence differences?
Well, I'd like you to explain to us what the tests are that are commonly used and what is it that they look at.
Okay.
Let me start here just for a minute.
There are two commonly used tests that look at sequence differences. And they are called DQA Alpha, and polymarker.
Let me start with the polymarker because it's a little easier. First of all -- yeah. Can you hang onto that?
It's abbreviated PM, standing for polymarker. This test looks at five different genetic locations, or LOSI. Five different locations.
Each of those locations has a name. But what I want to show you is that the results you get out are as follows:
There's two, two variations that a person could have. You could have a B at one location or an A" That is, you could inherit from one parent an A, and you could inherit from your other parent -- from your father, mother and father, in which case, the type that you would be would be AA. If you had inherited an A from one parent and a B from the other parent, you would be an AB. And if you inherit a B from both parents, you would be a BB.
And everyone is going to be one of these three types. Each of these variations is called an allele. You may have been familiar with ABO blood groups where the alleles are A, B, and O. Here the alleles are A and B.
For the polymarker tests, there are three locations that have this much variation where the types are called A and B and you can be an AA or an AB or BB. In this polymarker test, there are two genetic locations that the test looks at where you can be an A or a B or a C. And that ends up to give you the following types.
So that's all the possible combinations of an A, a B or a C. Each time inheriting one from one parent and one from the next.
Simultaneously, well, actually it's six because there's one location I didn't talk about and that's the DQ Alpha location.
The one you just did, yeah, we'll put a number on it. That will be 2182, I believe.
For DQ Alpha, there are a few more alleles than A, B and C and these have number designations.
So there's a 1.1, a 1.2, a 1.3 -- 1.3, a 2, a 3 and a 4. And if I wrote down all the combinations, if I remember correctly, it gives you 21 possible combinations and every one will be one of those 21 possible combinations of these six things.
And is this, once again, is an example of where you'll get one of those alleles from your father and one from your mother.
Yes. You might get a 1.1 from one parent and a 1.1 from another parent. Or you might get a 4 from each of your parents and be a 4, 4. Or you might get a 1.1 from one parent and a 4 from the other, in which case you'd be a 1.1, 4.
And again, this test, like the polymarker test, is testing for sequence differences at this particular location?
Yes. Each one of these that have a number designation in the DNA is different because there's a difference in the sequence of the base pairs at that DQ Alpha location.
Okay. Let's take a ten-minute recess, ladies and gentlemen. Don't talk about the case; don't form or express any opinions.
There are actually many more similarities than there are differences. At least 99 percent of DNA in humans is the same... However, a small fraction of the DNA is different and that's intuitively obvious because you can look around and recognize pretty quickly that people don't look alike unless they're related.
With the exception of identical twins. Identical twins look identical because, in fact, they have identical DNA.
In 1988, we did a large proficiency test sample set and got one incorrect match. And the same thing occurred in 1989.
Since 1953, yes, there have been many advances. We know a huge amount now that we didn't know -- well, I didn't know anything in 1953 about DNA, that is.