Thank you, ladies and gentlemen. Please be seated. All right. Let the record reflect we've been rejoined by all the members of our jury. Ladies and gentlemen, I apologize to you for the short delay, but I think you need to hear what's going on. And let's proceed. Mr. Clarke.
And is that one of the markers that you type samples at following the use of this PCR copying process?
DQ-alpha has six alleles. That is, there's six different forms of this DQ-alpha gene. And with that many alleles or forms, you can generate 21 different types in people. That is, so you're not confused, you--we each have two forms of any genetic locus or any gene. One comes from mother, one comes from father. So you're looking at how many combinations of these six things would exist, and there are 21 different combinations of the six alleles for this gene, DQ-alpha.
Is there a particular term used in science to describe what a person's type is when they have two forms of a particular genetic--added to a genetic marker?
All right. Well, let's start with that first. What do those terms mean? Are they--are they important? Do we need to know those terms?
Do you need to know them? Well, I don't know if you need to know them. Probably you do. When you have inherited from both parents the same form of a gene, that is so both your copy that you got from your mother and the copy you got from your father are identical, then you would be said to be homozygous, homo meaning the same and zygous referring to a zygote, which is combination of egg and sperm. So if you have two forms--you will always have two forms of the gene. And if they happen to be the same--let's use a very easy example. If you got a gene for blood group a from your mother and you got a gene for blood group a from your father, you would have two genes. Both are for blood group A, and you would be homozygous for that a.
Genotype simply--it just means what types do you have. Do you have an a and an a or an a and an B. That's your genotype, AA or AB.
As far as this marker, DQ-alpha is concerned, if everyone in the courtroom were tested, would each of us be one of these 21 different types or genotypes?
Does that mean because there are presumably more than--well, there are more than 21 people in this courtroom. Does that mean it's likely or necessarily true that at least two of us have the same genotype?
Well, probably for those genotypes or combinations of genes that are common, we'd find more than one person in the courtroom who had the same type.
That was actually going to be my next question. As far as these types that we each have, are they distributed absolutely equally or not?
I don't think we can--first of all, I'm not sure I can answer that question. That may be an evolutionary answer, and I'm not that knowledgeable about the DQ-alpha gene. However, what I know is that the types are not distributed evenly. So some types are very rare, some types are relatively rare and some types are very common.
We'll return to it later, but are there studies done to determine how common or how rare these individual types are?
There actually is a very large amount of work published on how common or rare particular DQ-alpha types are in many different racial or ethnic groups.
Still, with this DQ-alpha marker, how do you know it's a good marker to use in forensics?
Well, if you use as your Judge the first criteria that you want something to be relatively variable, that is a marker that shows a lot of variation is more useful than a marker that doesn't show very much variation. So on that criteria, DQ-alpha is quite good because with six alleles giving you 21 different types, that's a fair amount of variation and it's going to have useful information. The test that has been designed for forensic use for DQ-alpha, which uses PCR, only amplifies a very short section of DNA. That means the test is going to work well on samples that are degraded; and so for that reason, it's also very good.
Is it, for instance, more useful than the ABO blood grouping types that this jury has already heard about?
Well, with ABO blood groups, you have three choices of alleles. You can be an a--you can be A, you can be O or you can be B, and that gives you a combination of six different genotypes; that is, taking those three things two at a time. So that means one--each one of us will be one of those six types as opposed to DQ-alpha where each one of us would be one of these 21 types. So DQ-alpha is going to be able to distinguish us one from the other better than ABO.
Incidentally, of the six types for ABO you've described, are they actually type--fewer than that that can be actually determined from testing?
With normal serology testing, you can't distinguish all those types. People are developing DNA tests in which you will be able to distinguish all of those types.
All right. In addition to DQ-alpha, do you type other genetic markers following the use of PCR?
And are they collected--first of all, how many other markers do you type in the laboratory?
In our laboratory, we're typing--well, we're typing five other markers. Actually, recently, we've added three more, but they were not done here and we don't need to worry about those. So for purposes of this case, we typed five additional markers besides the DQ-alpha.
The five markers are collectively referred to as poly-marker or PM, and that's simply the name given to those--that's not a scientific name really. It's just the company who developed this test refers to it as PM, generally standing for poly-marker.
And are there different ways of describing those five different markers that are part of the poly-marker system? In other words, are they labeled by a particular name?
Yes. The five genetic locations that are tested in this poly-marker system, each one has a specific name.
Each one of them has a short abbreviation which stands for the longer name. If you just focus on the abbreviation, that's probably a lot easier. The five markers are LDLR standing for low density lipo protein receptor.
Okay. The other is GYPA, the third is HBGG, the fourth is d7s8 and the fifth is GC.
Now, of those five you just described, one of them had that notation of the letter "d" with a number; is that right?
Would that then be another one of those markers where its function in the body is unknown?
The other four are actually genes and they have--the letters are just an abbreviation for the name of the protein that's been very well described for that gene.
Now, this set of five markers that are part of poly-marker, how do you know they're good to be used or appropriate to be used in forensic work?
Each one of these markers doesn't--does not have a lot of variation, but grouped together, it still is informative. And really, the answer to your question is, in addition to the theoretical idea of how informative is this marker, this test has been designed to be also useful specifically for forensic purposes. The sections of DNA that are amplified are small and the typing is on the dot blot strips that you saw in the tray a while ago. So it's a test that's easy to do in the laboratory and it works on a--and has been shown to work on a very wide variety of samples. So it's applicable to all kinds of samples that come in in terms of forensic casework.
Incidentally, that tray with the strips that you identified and was shown to the jury, was that actually a poly-marker set of typing strips?
Yes. The strips that were in the tray when you looked at them have the five poly-marker loci. And if you were looking at the strip, you would see that there were letters on the top of each one, and it's the LDLR, GRPA, indicating the dots in that set of the--in that portion of the strip or for that particular genetic location.
Going back to DQ-alpha for a moment, how long has it been used in forensic casework?
Going forward then to the poly-marker genetic markers, when were they begun to be used in forensic casework?
We started using the poly-marker test in January of 1994. There probably are some other labs who were using it earlier than we do, but I don't know exactly how--it wouldn't have been a lot earlier. Somewhere maybe within a year or so before we did. So let's say it's been being--it's been used in forensic casework since approximately 1993.
Is there some process that these genetic markers undergo before they're actually used in casework or did someone just discover a gene and start using it immediately?
There's actually a very--what usually ends up being a very long process of development of the marker and then what's referred to as validation. That is, if another laboratory uses a marker, that doesn't necessarily mean that I can just bring it into my lab and start using it. So each lab that uses a particular test generally goes through a set of experiments in their laboratory to make sure that in--in the hands of the people in that laboratory, the test functions as it's supposed to, it meets its specifications and that everybody that is using it understands what it is they're doing. So there's development of the marker, then there's validation of the marker, which may occur in many different labs, and then there's training the staff in a particular lab to use that--that marker.
And do you ensure that that marker can be correctly typed in evidence type samples before you even begin casework use of them?
As far as these poly-markers--well, let's start with DQ-alpha. Is DQ-alpha a marker that's tested--and I think you said more than one laboratory uses the poly-marker system. What about the DQ-alpha system?
The same is true of the DQ-alpha system. It's used by many forensic laboratories around the country and really around the world.
That was going to be my next question. To your knowledge, are the poly-marker and DQ-alpha genetic markers used worldwide?
You touched a little bit about as far as the five poly-marker genetic markers, that their variation is not as great as DQ-alpha?
Without getting into each one individually, can you tell us briefly how much variation that each show?
The five locations that make up the poly-marker system, all of those five locations either have two or three alleles or forms and they're all--they--they all are designated a or B, and if they have three forms in the kit, they're designated A, B or C. So you can--for example, if you have a marker that has an a type and a B type, any given individual then could have two A's, they would be an AA, they could have two B's, they would be a bb or they could have an a and a B and they would be an AB. So if you have two alleles, the most number genotypes you can have is three.
DQ-alpha and these poly-marker genetic markers, do they represent sequence differences or length differences as you described them earlier today?
As far as the actual typing of these DQ-alpha and poly-marker genetic markers, do you actually conduct these tests from scratch in terms of materials and what you use to be able to type samples?
These tests, the DQ-alpha and the poly-marker, come as a kit, which means, you're buying in a box all the things that you need or almost all the things you need to do the test. Actually, you'd end up making a few solutions in the laboratory. But the strips come in the kit, the chemicals that you need to do the reaction, to create the blue dots come in the kit, the tray comes--you have to buy the tray and the kit. I guess actually you buy them separately. And the PCR mix that contains the primers and the polymerase and the a', G's, T's and C's. All that comes in the kit. So you're just supplying some very simple to make solutions and using the kit to do the test.
When you conduct the more powerful RFLP typing technique, do you use kits to do that?
Why is there a difference between the two approaches to DNA typing as far as kits are concerned?
Well, somebody took the time to make the kit for DQ-alpha and poly-marker and nobody has tried to market a kit for RFLP testing. I guess you could, but it would have an awful lot of components.
It's a good test and it's available and it's had an enormous amount of work done on it. If it wasn't a good test, you wouldn't use it. If it wasn't available, obviously you couldn't use it. So the company that makes the kit has put a lot of work into it and it happens to be very, very good.
All right. Turning your attention if I can to the last genetic marker that we will talk about, d1s80, does that represent a genetic marker that's typed based on length differences as opposed to sequence differences?
Was DNA--I'm sorry. Was d1s80 developed as a marker to be used for forensics or did it have some different origin?
As far as I know, it was--it wasn't discovered--I mean it was discovered, but then basically it was developed specifically for forensic use. I really don't know if there's any research use for d1s80 or not.
We've run this marker in our lab. We're not currently using it in our routine casework, but we've run it in our lab, and I've also actually read a number of papers that had to do with d1s80 and heard a lot about it at various scientific meetings.
The reason we're not using it in our casework is not because it's not a good marker. It's a very practical reason; and that is, the kind of gel that the d1s80 is run on is not a kind of gel that we would run other markers on. And so essentially, we aren't using it because it's a fair amount of work to do it and you get one piece of information off, that is the d1s80 piece of information. There are some other markers out there that we chose to use instead. They're not quite as informative as d1s80, but I can do more of them at a time. And so it was a very practical decision that you would just make in the lab to say, "do we want to do test a or test b?" It has nothing to do with whether or not d1s80 is a good marker. It was just our choice to go with another type of marker.
So there are practical considerations that go into each laboratory's decision of what markers they're going to test?
That's right. And because not every--not every laboratory that does casework has the same type of casework or the same casework demands. And so all of those things go into your decision about what markers you're going to be using.
As far as your familiarity with this d1s80 marker, what type of variation does it show?
The d1s80 has over--at least 24 alleles. That is at least 24 forms. I don't--haven't calculated how many genotypes that could give you, but you can imagine 20--at least 24 things taken two at a time. You can have many, many, many combinations. So it's a very informative marker and it's--except for the gel system, which takes some pains to do, it's a very good one.
All right. I would like to shift topics if we can and ask you, are you familiar with the term "contamination"?
It means so many different things that maybe I should try to break them down in terms of what it means to me.
If you think of a biological sample, say you take a blood sample from someone in sterile conditions, you have a very clean sterile sample. That's sort of the ultimate in a good--in terms of having a good sample. As soon as you take that sample out of a sterile condition and say you have a bloodstain on a piece of cloth and you could think about well, that bloodstain is no longer sterile and it could be, quote, contaminated by anything that's on that cloth. So that would sort of be a second or let's say a first level. Now, in terms of analyzing evidence from a crime scene, you're always getting things that are like that. You're never being presented with a sterile sample. So in terms of a crime scene, contamination would mean that the sample is as it--that is, in retrieving the sample, it is as it was deposited, that nothing's been added to that sample in the process of picking it up, taking it to the crime lab and so forth. And then you can also say, "well, I have my sample from the crime scene. It came to the crime lab in exactly the same condition that it was at the crime scene." And then clearly, you need to be concerned with whether or not in the process of working with that sample in the laboratory, are you introducing any contamination from any source in the laboratory. So you can't use it very well as a generic term. I mean, you could, but unless you specify what kind of contamination you're talking about, then it becomes very difficult to discuss because you can--you can break it down and you can have many different kinds of contamination, some of which are inherent in the fact that you have a piece of evidence and not a sterile blood sample and some of which are not inherent and that could occur along the way in how that sample is handled.
The idea of analyzing a piece of evidence is that you learn something about that piece of evidence. You're not interested in learning about-- that is, if you contaminate that piece of evidence with--let's talk about, say, you contaminate a piece of evidence with some other biological sample or some DNA in the laboratory. You--that will then interfere with your being able to come to a valid conclusion about that piece of evidence. And so you would certainly prefer not to have anything interfere with coming to a valid conclusion about a specific piece of evidence.
As far as this area of contamination--and you've described the two basic DNA typing approaches that you use, both RFLP and PCR--is your concern about contamination the same for both of those techniques or is it different?
The PCR test is much more sensitive. You can do a PCR test with a very minute amount of starting material. So you would be more concerned. It doesn't mean you're not concerned with contamination for RFLP, but you're going to be more concerned with contamination when you're dealing with doing a PCR test on a piece of evidence.
Okay. Let's take RFLP to start with. What steps if any do you take to deal with the problem or the potential problem of contamination in that testing process?
Okay. And what I'm--my answer is going to then be what we're doing in the laboratory because we're simply receiving a piece of evidence from outside. That is, we're not collecting the evidence ourselves. In the laboratory, our work is done in a--it's called a biological safety cabinet, but basically it's a setup where you have a piece of glass and you have a working area and the air is circulated within that working area to both protect what you're working on and to protect yourself. So you have some containment in the area that you're working with these samples. In our laboratory, when a sample is moved from one tube to another, that is, the tube has a label, it has a case number label and a sample number label, when it's--and in the process of doing DNA extractions and doing this testing, there are points where you have to remove the sample from one tube and put it into another. That transfer--the labeling on the tubes is witness. That is, somebody is checking to make sure that if I'm transferring from a tube that's marked 02, then I'm transferring it--the second tube I have in my hand is also marked 02. The other main precaution is that we do the DNA extractions for evidence samples as opposed to known samples. That is, when I say known, I mean you have a standard sample from a known individual. Those extractions are not done out in--well, they're done in this hood, but they're not done at the same time. So the knowns and the evidence samples are not worked on for purposes of DNA extraction at the same time.
So those are the primary steps undertaken with regard to the potential for contamination as far as RFLP typing is concerned?
What about PCR? Do you undertake any different steps or additional tests to deal with this potential problem?
There are many additional features of how you do a PCR test to deal with minimizing any problems with contamination.
Could you describe those, and are there general categories that you can place them into as far as the steps that you take?
Okay. The--let me think about this a second so it comes out organized. The PCR--the DNA extractions for PCR are done in a separate location than the extractions for RFLP. Now, in our lab, it happens to be that there's a separate one of these biological safety cabinets that's only used for PCR extractions. And the reason for that is that if you're doing something--if you have enough DNA to work with RFLP, that's a fair amount of DNA compared to what you might have in a PCR sample. So we don't want to be handling the large quantities of DNA that you can--that you would have for RFLP in the same place with the same equipment that you're handling the very small amounts. So most laboratories will have a separate location to do DNA extractions when those DNA extractions are for PCR. And in our lab, it's a separate--one of these hoods. The sample is--and in addition to that, for example, in the hood, the pipette-man or the, you know, the piece of equipment you're using to add things to the sample and take things out of the sample are specified. That is, you have a pipette-man that's used only for DNA and you have a series of pipette-man's that are used for everything else, so that you're not interchanging the piece of equipment that you're using to handle the DNA. You are that concerned. And the tips that you're using for the pipette-man are these art tips that have a filter in them so that you can't get any part of your sample up into the pipette-man and, therefore, accidentally transfer it to the next thing that you work with.
Well, basically that's--everything I just said relates to the area where the DNA is extracted.
All right. Extraction was the first basic phase of dealing with evidence for purposes of PCR typing; is that correct?
Earlier, you described the second major phase involves what's called amplification, this copying process.
Do you take any precautions or additional steps to again deal with the potential for contamination during this particular phase?
Well, first--now that you've finished the extraction, you want to set--you're going to set up the amplification. That is, you go through that process where you have a tube, and in that tube, you put your DNA and you put all the things you need to do the PCR, the primers, the polymerase and so on. That set up is done in a second area, a very clean area, and it's--in our lab, it happens to be a very small room, but it could--it could be some other just separate location. So the set up of the PCR reaction is done away from where the DNA extraction is done. Once that reaction is set up, it's then carried to yet a third location where the thermal cycler is where the amplification takes place. Once it's gone into that area where the amplification takes place, it does not ever go back in the other direction.
I think about--in terms of the PCR, you have started with a tiny amount of material, and now after the process of amplification, you've generated millions of copies of that tiny amount of material. The biggest contamination problem that can occur with PCR is transferring some of that amplified product back to where you're starting out with, because that amplified product will amplify really well if it gets into anything else. So the--it's not the only concern, but the biggest contamination concern is that you do not get any of that amplified product, which is now a lot of DNA, back into anything that you're ever starting out with. So the DNA samples go from the extraction area to the set-up area, to the area where the thermal cycler is and where you then do the analysis of the amplified product and they do not go back in the other direction.
And that's to, again, avoid this problem of lots of DNA being mixed in or commingled with small amounts of DNA?
Now, as far as--and I believe you described the fact that evidence is not extracted at the same time as known samples or did you mention that?
I did mention that with regard to RFLP testing, and the same--that same general procedure. That is, you don't do the DNA extractions for evidence and knowns at the same time. That's just a general precaution we use in our laboratory, and it's applied to everything.
So as far as RFLP typing, your primary steps to avoid contamination are extracting samples at a different time, that is knowns versus evidence; is that right?
You utilize those same two precautions and the other long list of precautions that I just talked about.
As far as this case--and you described a little bit about the amplification phase. Was it correct that evidence samples in this case were amplified or did it happen that they were amplified on a different date from known samples?
In this case, it happened that that's the way the testing was done. Not only were they, the DNA extractions, done at a different time, but the amplifications, that is putting the tubes in the thermal cycler and allowing the amplification to take place, that also was done at a different time for the knowns and the evidence samples. That's not necessarily the--the way every case is worked. It just happened to be the way this case was worked really based on when we received samples and when we did each one.
As far as these precautions--and let's focus on PCR for the moment. The precautions that you've described that you use in the laboratory, are they unique to forensic science or are they used in other areas of science involving PCR?
They are definitely not unique to forensic science because every lab that's using PCR, no matter what the reason, is still doing the same thing. They're taking a small amount of DNA and making millions of copies of it. So any laboratory that uses PCR for any reason has got to be concerned with making sure this amplified DNA that they get at the end doesn't get transferred back to any--any place along from the start of the sample on forward.
Exactly. And I'm not--I'm not--I don't mean to imply that, for example, a research laboratory would take exactly the same precautions that we would. They may be slightly different. But no matter what the setting is for PCR, you have to be concerned about making sure amplified product doesn't get back to your starting point.
So in terms of these--and just in terms of summarizing, these precautions that you take for PCR, they include the way you handle evidence; is that right?
The way you extract evidence in terms of when it's done at a same or different time than known samples in a case?
As well as the direction of flow so to speak of a sample going in one direction only as you described and not backwards?
As well as--and incidentally, are there other steps taken that to your knowledge we'll discuss later as well in terms of dealing with the potential problems of contamination?
Now, I'd like to shift topics a little bit to specific forensic case samples that are encountered. And in particular, with regard to evidence, can evidence at, for instance, crime scenes be subjected to basically the environment and the elements in that environment?
I don't think it's a question of "can." I think it's a question of that's absolutely bound to happen.
Okay. What types of things can happen, for instance, to a piece of evidence found outdoors?
Well, it's going to be exposed to whatever temperatures are outdoors, whatever lighting conditions. There may be a lot of sunlight on that day. Could get rained or snowed on. I mean, whatever--if it's outside, it's going to be exposed to whatever's going on outside from the time that it was left there until the time that it's picked up.
Incidentally, would the same be true, for instance, of a soldier killed in battle, out in a battlefield?
You've described a little bit about, for instance, heat. Would that represent one instance of an environmental effect or something that's possible that happens outside?
Well, I mean, you said outside. So I said, well, it could get rained--I mean, in California, maybe not, but--
With respect to these influences or effects like heat, humidity, rain and so forth, what do they do to DNA? What's their effect on DNA?
Basically, all of those things over time will act to gradually degrade the DNA; that is, break it up. The generalization that--that most everybody is aware of is that dry and cold works to preserve DNA and warm and moist works more towards degrading DNA. That's just a generalization, but that's pretty much the case.
We'll talk about storage of samples in just a few moments. But is there anything about these environmental effects, whether sunlight, humidity, rain, et cetera, that can actually change DNA from one type to another?
Well, you know that at a relative--at a very basic level because these things will not change the DNA sequence. Light, heat all of these things will not result in changing the DNA sequence. If you think about the kinds of locations that we're testing, we're testing lengths of DNA. Suppose you're doing an RFLP test and we're using my DNA and I have a DNA fragment that's 5,000 base pairs long. Degradation of DNA is a random process. There's no method or no enzyme that can go in from an environmental perspective and say, we're going to take that 5,000 base pair piece and it will then be converted to a 3,000 base pair piece, just a nice clean thing, goes from 5,000 to 3,000. That isn't what happens. Degradation is random. So that 5,000 base pair piece as it degrades is going to be cut up in many different places. And so it just gradually gets smaller and in many different sizes. So there is no environmental force, there is no environmental effect that can work to simply change one type and make it become another. You may lose the type altogether. You may degrade the DNA so much that you can't type it. But you won't just change types from one to another. Doesn't happen.
Let's talk a little bit about storage. And you mentioned briefly about drying and--I'm sorry--making a sample cold as being a means or a method to preserve DNA?
Problem with moisture--with high temperature and high humidity is that that enables bacterial growth, and bacterial growth will result in the DNA becoming degraded. That's generally the problem with high temperatures and humidity. Dry conditions and very cold conditions inhibit any kind of bacterial growth. And so they tend to be very good for storage. And keep in mind, unless your sample is sterile, sterile meaning the presence of no bacteria whatsoever, then any moisture and any heat will promote bacterial growth and that bacterial growth will gradually degrade the sample.
What if you don't, for example, refrigerate or freeze a particular sample of DNA? What happens?
Well, it sort of depends on what stage it's at. If you're just at the evidence stage where you have some stain on some piece of clothing or some other substrate, then you really will see different effects with moisture and heat. Once your DNA is extracted--and it's really very clean. You don't have bacteria in there anymore. They've been destroyed in the extraction process. You may have bacterial DNA, but you don't have living bacteria. They've been destroyed. And so once your sample of DNA is purified, it does store over time best if it's kept cold. It's usually stored at minus 70 or minus 20 degrees centigrade. But if you left it out on the bench top at room temperature, you could really leave it out there for a very long time before it would be useless.
Your work in the laboratory includes the receipt of certain--paternity samples for paternity tests; is that right?
We receive samples for paternity testing usually by federal express, and they're usually drawn from the individuals that are going to be tested the day before, they're packed up in a Styrofoam container so they can't break, they're shipped at ambient temperature, whatever temperature the air is, and received by us usually about 24 hours later.
Are these shipped--now, you mentioned the term, they're stored at ambient temperature, whatever temperature the outside is?
Well, that is, when they're given to federal express. Federal express isn't putting them in a refrigerated vehicle. So they may be flown to us or come in a truck. But whatever temperatures happen to be in that truck or the plane or, you know, all the various things it's going into before it's delivered to us, that's the temperature they're at.
What happens, for instance, with one of these liquid blood samples? Well, let me rephrase that. Do you in the course of testing even in your forensic casework, that is identification casework other than paternity, do you receive samples in a liquid blood form?
Are they maintained in any condition as far as refrigeration in terms of their shipment or what they're shipped in?
If we're receiving a liquid blood sample for a forensic case, it's usually shipped in the same manner. Occasionally it's hand-delivered. Mostly, it's sent by some kind of carrier like federal express.
What can happen to these liquid blood samples? What do you see in your casework that can happen as far as the ability to type DNA?
Well, it makes a big difference. If we're talking about liquid blood samples drawn from a known living individual, they're usually drawn in the same way the paternity samples are shipped to us and they're just fine. There are other types of blood tubes that you can draw people's blood into that have other types of preservatives other than EDTA. Those other types of preservatives are not as good for maintaining the condition of DNA as EDTA is. That's absolutely the--that's the preferred way for us to have a blood sample.
Well, I was just going to say, if the blood sample's from someone who's died, then you would be more concerned to make sure that it had been cool--it had been stored properly and that it had been drawn in an EDTA tube.
Is that because blood taken from dead bodies is subject to this degradation process in a much faster fashion or faster fashion than blood from a living individual?
If blood is degraded in a liquid form, whether it's from a sample from a Coroner's office or paternity sample or another sample that you received in liquid condition, is there anything about that degradation process that can change the types of the sample involved?
What happens when it degrades? Is it the same as happens to, for instance, a stain that you described maybe outdoors?
Now, as far as other things that exist out in the outside or in the environment, are there any in particular that can create a problem in your being able to type the DNA in that sample itself?
Any sample that is sufficiently degraded can be so degraded that you can't type it by any of the typing methods that are currently available. And there's an exception to that. It's typing of what's called mitochondrial DNA, which is DNA that is not in the nucleus. And that is the absolute last resource. That is, if the sample is in such bad condition, you can't do anything else with it, you might be able to do mitochondrial DNA, and there not very many places that will do that, that have that capability.
As far as what's out there so to speak in the outdoors, what about soil? Does it play any role in the ability to type DNA?
Samples that are retrieved from soil are very difficult to type. Frequently you do not get any result.
Presumably, you have bacteria in the soil that are participating in degrading the DNA. However, I don't know of a definitive experiment that's shown specifically that it's bacteria. So I only can tell you because this is the common experience, it's in the literature and many labs have had this experiences, if they are taking a blood or semen stain from soil, their success rate with those samples is very, very low.
Is there anything about the presence of soil or leaves that could change the types that are found in a particular DNA sample so that they would be typed differently from what the contributor of that DNA actually is?
As far as laboratory precautions that you take again--and we're--and let's focus, if we can, on PCR typing, the precautions that you described earlier--is it a danger, for instance, if someone's coughing around a sample? And let's refer to an analyst.
Okay. So what you're asking me is, if I have a piece of evidence and I'm going to do PCR analysis on it and somebody's coughing, could that present a problem?
It's possible. It's not--I don't think it's real likely, but I can't tell you it's impossible.
Well, if you make it generic and say if I as the analyst transfer any of my cells onto the sample, then I could create a problem with that sample. But you have to think how much material you're transferring to that sample relative to how much material is there in the sample already. So it's--there's not a very--there's not a black and white answer to that question and there may not be a simple answer to that question. I--there is an experiment in the literature where they actually tried to contaminate a sample by the way the sample was handled, and it didn't cause any problem. That doesn't mean that it would never cause a problem. So you do want to be concerned about it, but you shouldn't assume it will cause a problem absolutely every time something like that occurs.
In particular, are you referring to a publication in the scientific literature directly addressing the potential for problems as a result of some of these items we just discussed?
And did that publication specifically deal with this question of whether or not these various influences could affect the ability to correctly type DNA samples?
As far as your reading of that publication, did that lead you to render any conclusions in your own mind about the appropriate way to deal with samples that are obtained, for instance, at crime scenes?
Did it corroborate what your opinion was before you had even read the publication?
Well, at the time that I read the publication was during the time we were just starting PCR. So it added to my thinking, which was sort of still being formulated.
Incidentally, as far as a stain let's say at a crime scene, to your knowledge, is there any reliable method of determining how old that stain is?
As far as these environmental influences--actually, let me rephrase that, if I may, your Honor. As far as these items that we discussed, touching--well, sneezing, coughing, et cetera, do you have a personal opinion about their impact on your ability to properly type DNA samples that have been subjected to PCR amplification?
With regard to these various impacts--well, let me rephrase that if I can. As far as your own experience in the laboratory with testing samples--and I'm referring to evidence samples--has that included samples obtained from a variety or under a variety of different circumstances?
How long have you been engaged in that? How long have you been in the laboratory gaining this type of experience?
Well, when I--when you say "you," really we're referring to my whole lab staff. Since about--well, since before 1992. We did a lot of work. We did a lot of validation work before we actually began to use PCR in casework. So we have the experience from the validation work and from the casework.
As far as in the laboratory--and I'm going to direct your attention in particular to sample handling; for instance, extraction, amplification and typing of DNA--have you examined the results from your casework since you've used PCR in your laboratory?
Do those results include certain steps to determine if in fact the process of using PCR and then typing it is leading to or is actually including foreign DNA being typed in samples? Is that question clear?
Excuse me. I still have an objection both as to vagueness in the question and secondly, again, foundation because we don't know whether it's to samples that are coming in--
Sure. As far as this actual PCR typing process that's gone on in your laboratory, do you take steps to detect whether or not there may be DNA present that you're able to determine types from that didn't come from a particular evidence sample?
A control is some kind of sample that you use alongside your other samples or at some point in the process, again, to help you determine whether or not your process has worked correctly and whether or not you have a valid result.
All right. We'll return to the nature of the controls that you utilized in the laboratory a little bit later. But do those controls provide you with an opportunity to determine whether or not foreign DNA; that is, DNA that's not part of the sample, is being injected into the process?
All right. And has that gone on since DNA, that is PCR type DNA testing has gone on in your laboratory?
With regard to these potential influences like coughing, sneezing and touching, do you have an opinion about your ability to detect them as far as if they have any impact on your typing of evidence samples?
With regard to handling the samples from the point of the extraction forward, the controls will allow you to determine whether anything that you're doing has had an impact on that sample. Obviously we can't make any determination regarding what's occurred to the sample before it comes in our laboratory because then the sample is as it is. We don't have any experience in our laboratory of picking up in a sample control the types of the analysts who have handled that sample.
So it's been your experience in your laboratory that these considerations, these things that can happen simply don't happen in your laboratory?
It's been our experience that we are not detecting them happening in our laboratory. As far as we can tell, they are not impacting on the analysis from the point that we get the sample.
All right. Ladies and gentlemen, we are going to take our recess for the morning. Please remember all of my admonitions to you; please don't discuss the case amongst yourselves, don't form any opinions about the case, don't conduct any deliberations until the matter has been submitted to you, do not allow anybody to communicate with you with regard to the case. And we'll stand in recess until 1:00 o'clock. All right. I would like counsel here promptly at 1:00 o'clock. All right. And, Dr. Cotton, you may step down.
There is no environmental force, there is no environmental effect that can work to simply change one type and make it become another. You may lose the type altogether. You may degrade the DNA so much that you can't type it. But you won't just change types from one to another. Doesn't happen.
Because it's there.
Once your sample of DNA is purified, it does store over time best if it's kept cold... But if you left it out on the bench top at room temperature, you could really leave it out there for a very long time before it would be useless.
The biggest contamination problem that can occur with PCR is transferring some of that amplified product back to where you're starting out with, because that amplified product will amplify really well if it gets into anything else.