Generating forensic DNA profiles (HD version)


Hi, Iím Dan Krane. Iím a Professor of Biological Sciences at
Wright State University in Dayton, Ohio, and Iíd like to talk with you for the next 50
minutes or so about how it is that you go about generating forensic DNA profiles. If youíd like to see this PowerPoint Presentation
on its own, you can find the file associated with it at www.bioforensics.com. And youíll also be able to find there quite
a few additional materials that might give you a greater understanding of what it is
that Iíll be talking to you about in the next short while. But with that out of the way, letís just
get right to work and talk about how it is that crime laboratories generate DNA profiles
for use in courts, both within the United States and around the world these days. Perhaps the best place to start is just with
a quick overview of how it is that DNA tests have been generated for the past 20 or so
years since DNA testing first appeared in criminal courts. There have essentially been three different
types of DNA tests that have been used over the course of the past few years, 20 now,
for generating DNA profile. In the very start the first DNA tests were
generated with a methodology thatís hardly ever seen these days except in post-conviction
appellate cases; itís a RFLP based test. Itís a restriction fragment length polymorphism
type of test. The type of evidence that was generated and
shown to jurors was called an autorad, or an autoradiogram and it looked like these
bar code patterns that you see on this particular example. And here the relevant bit of information was
associated with where these bands moved, thatís what told us if an allele was present or not. And donít worry if youíre not sure what
an allele is, Iíll be defining that sort of thing for you in just a few minutes. But thatís what the original DNA tests looked
like back in the late 1980ís and early 1990ís. These tests took about a month, sometimes
two or three months to generate results, and they needed a blood stain about the size of
a quarter to be able to give rise to these types of results. Took a long time; nowadays, at least in comparison
needed a large amount of starting material. And so there was clearly some room for improvement,
and essentially that left the door open for a second generation of DNA tests. These are the very first PCR based tests. Again, donít worry if youíre uncomfortable
with what PCR means, weíll be talking about that more in just a few minutes. But these very first PCR based tests were
called DQ Alpha, or polymarker tests. Here the critical bit of evidence was coming
not from an autoradiogram, a piece of x-ray film, but rather from a series of strips known
as test strips. And what we were looking for here is the presence
or absence of these blueish dots. You can see in some spots the blue dot would
appear and other spots they donít appear. The particular pattern of dots would give
us some insights in terms of what sorts of DNA molecules were present in a particular
sample. Hereís the thing ñ the first generation
of DNA tests took about a blood stain about the size of a quarter to generate results,
would take months. These second generation tests could work with
about a hundredth of that amount of material; blood stains that were almost too small to
be seen with the naked eye and results could be generated in the span of an afternoon,
so much faster and much more sensitive in terms of how much staring material was necessary. But thereís a third criteria by which we
judge DNA profiling tests ñ that third criteria is discriminating pallor. And so while the first steps werenít very
good in terms of their sensitivity, and they werenít very good in terms of how quickly
results were generated ñ these first two criteria ñ they were very discriminating. And it was possible to assign statistics in
the ballpark of one in a million or one in ten million as the chance of a coincidental
match between a suspect and the true perpetrator of a crime, if the suspect wasnít the source. The second generation tests did very well
in terms of sensitivity and in terms of the time needed to generate a result. But they didnít deliver as much in terms
of discriminate pallor. And statistics would frequently be described
in terms of one in a few hundred or maybe one in a few thousand. And that in turn left open the opportunity
for a third generation of DNA tests, these automated STR tests. Theyíre hitting a homerun on all three criteria. Here DNA profiles can be generated with samples
that are literally described in terms of cells, in the ballpark of 100 cells is sufficient
for most of the commercially available kits to generate a DNA profile; much less than
what could be seen with the naked eye. In fact, verging on what it is you can see
with a microscope. And theyíre also really fast, from beginning
to end it takes just a few hours to generate one of these types of DNA profiles. And theyíre really hitting a homerun in terms
of discriminating pallor. Youíll see that these types of test results
are often associated with statistical weights described in terms of quintillions and quadrillions,
literally billions of billions is the chance that a sample might coincidentally match a
randomly chosen unrelated individual. So this is still state-of-the-art to this
day. They became popular tests in the mid-1990ís
and they sure look as if theyíre going to remain popular for at least the next 5 to
10 years before they get supplanted by a new methodology. STR tests, or automated STR tests are what
it is that Iíll be talking with you about how theyíre generated for the rest of this
particular video, and itís also going to be the basis for all the other discussion
weíll have for the following videos in this particular series. But again, thatís state-of-the-art for today. Weíll talk about how it is we get those automated
STR test results as we move along here today, but before we do that, let me also draw your
attention to two newer kids on the block so to speak, as far as DNA testing goes. These two other tests have been increasingly
popular and increasingly commonly used in forensic investigations. They are mitochondrial DNA testing and YSTR
testing. Mitochondrial DNA testing ñ these test results,
these tests are actually more sensitive than the automated STR tests. The principle reason for that is that while
every cell contains within it two copies of all of our genetic material ñ one copy we
get from our mother and one from our fathers ñ each cell that has one of those nuclei
will also have associated with it thousands of mitochondria. And each mitochondria will have within it
multiple copies of whatís known as mitochondrial DNA. The thing is the mitochondrial DNA is relatively
small compared to the DNA thatís present inside of the nucleus, so thereís not as
much information there for us to look at. But it is present in very high copy number,
and so it gives us the chance to even get results from a tiny fraction of a cell. The other drawback with mitochondrial testing
is that all mitochondria are maternally inherited. And what that means in simple English is that
an individual inherits all of their mitochondria from their mother. And her mother in turn got all of her mitochondria
from her mother. That in turn causes the tests to be very sensitive,
but not as discriminating. Because we know when we find one particular
kind of mitochondrial DNA that there are probably many other maternal relatives at the very
least who would have the very same mitochondrial DNA. In other words, mitochondrial DNA sequences
arenít likely to be unique within the broader population. The other new kid on the block are YSTR tests. There are some parallels between YSTR tests
and mitochondrial DNA tests. The YSTR tests are paying attention just to
the STR markers, the particular locations that reside on the human Y chromosome. And Iíll tell you what, you probably donít
need a biology professor to mention this or to explain this to you, but I can say with
some authority that men and women are different and the basis for the difference is ultimately
in their DNA. Women, I think we all appreciate, have two
X chromosomes; men have an X and a Y chromosome. Thereís information on that Y chromosome
that we can look at. And when we think that thereís evidence,
that thereís a male contributor to an evidence sample, YSTR testing is something that might
give us some insights in terms of what that maleís DNA profile looked like without being
distracted by things that may have been contributed by a female. That can be a very helpful feature when weíre
talking about a mixed sample, such as those that might come from a rape investigation
where you have a large amount of female DNA mixed with a small amount of a perpetrator
males DNA. But I said there are parallels between the
YSTRs and mitochondrial DNA testing, here they are. Whereas mitochondria are exclusively maternally
inherited, Y chromosomes are exclusively paternally inherited. I got my Y chromosome from my father, he got
his from his and his fatherís father got his Y chromosome from my great grandfather. Thereís a reasonable expectation that all
four of those individuals, myself and those three progenitors of mine, would have exactly
the same YSTR profile. So, the paternal inheritance causes us to
have some concern about just how discriminating YSTR test results are, but again, they can
be very useful when weíre talking about a mixed sample where there may be a preponderance
of a females DNA and we just want to get a good look at what was contributed by a male. But enough about those two new kids on the
block ñ mitochondrial DNA testing and YSTR testing ñ letís get to work talking about
the automated STRs. To do that I have to start by talking about
some background material; I want to get some definitions out of the way so that later on
I can just use the jargon and the shorthand and not slow down to explain it as we go. So the next couple of slides are going to
be talking about just some vocabulary terms. Letís start by talking about the kind of
quantities of DNA that are necessary to generate a DNA profile. The bottom line is not very much. Weíll cut right to the chase with this particular
slide; our bodies are made of trillions of cells. We leave cells behind in things like fingerprints
all the time without thinking about them. The kind of DNA that you leave behind in a
fingerprint is plenty enough to generate a DNA profile using widely available commercial
DNA testing kits. Most kits recommend that the starting material
correspond to the amount of DNA that you would get from as few as 100 to 200 cells. And again, thatís about the same number of
cells that you might find associated with a typical fingerprint. So the bottom line here is we donít need
a lot of material to start, and we can get it from just about anything thatís come in
contact with an individual. But it raises the specter of just what a picogram
is. Iíve said that the typical cell has in the
ballpark of six to seven picograms; the minimum amount thatís recommended by the manufacturers
of the test kits that are used is about 500 picograms. Letís talk just what that means in plain
English. From our day to day lives we know that a gram
of sugar is about a quarter what it is youíd find in a sugar packet, like the ones you
might get at a restaurant over the course of your lunch. So a typical sugar packet weighs four grams. A quarter of that is one gram; thatís a quantity,
a weight that I think weíre all very familiar with from our day to day lives. A single crystal of sugar from within that
packet is typically about a millionth of a gram. And if you took a millionth of a gram, or
a milligram and you split it up a thousand times you would find that youíre talking
about a nanogram. So a nanogram is actually a millionth of a
gram; itís a thousand thousandth. And if we took that millionth of a gram, a
nanogram and we divided it up a thousand times again, what weíll find is that now what weíre
talking about is one of those picograms. In other words, a picogram is literally one
billionth of a gram; one millionth of one of those specs of sugar that you would find
coming out of a sugar packet. In normal human terms, an unimaginably small
amount of starting material, but for DNA tests thatís plenty enough to get us started. Now at the same time that we talk a lot in
DNA testing about picograms, Iím also going to be using volumes. Picograms talks about mass, about weight;
volumes we need to talk about microliters, or millionths of a liter. So this next slide gets us a little bit of
an insight, a worldly sort of perspective on whatís going on with a microliter. I think we all have experienced dealing with
two liter bottles of soft drinks. If you cut that in half then obviously weíre
dealing with a liter. If you took just a thousandth of a liter you
would find that you get a milliliter. And in practical terms thatís about the same
amount of liquid that you would use to fill a thimble, maybe a smallish thimble. So a milliliter is a thousandth of a liter. Interestingly, chemists will often have a
precise meaning when they say ìadd a drop of something to a solution,î a chemist is
often trained to know that that corresponds to 100 microliters, and a microliter is actually
one millionth of a liter. So a microliter is something that you can
see with your naked eye, but itís awfully small. So, when a molecular biologist is talking
about DNA, in that context weíre usually also talking about picograms and weíre talking
about microliters. And for better or worse to follow whatís
going on with DNA testing we need to be comfortable with those terms. Bottom line, a picogram is a billionth of
a gram and a microliter is a millionth of a liter. Now that weíve got those vocabulary words
under our belt, let me move on to talk with you about a few other very basic vocabulary
terms that molecular biologists use when theyíre talking about DNA testing and forensic DNA
profiling. So letís start with talking about what is
a DNA polymorphism. I understand the word polymorphism may seem
a little bit unusual and intimidating at a first glance, but itís really very straightforward. The translation here is simply this ñ ìpolyî
is the word that we use for ìmanyî and ìmorphismî is ìmorphî is ìform,î so what weíre talking
about here with a DNA polymorphism is quiet simply a region of DNA thatís very likely
to differ from one person to another. And why does it differ from one person to
another; simply because the DNA in that region is likely to come in many different forms. Literally, later, youíll see in many different
sizes, but for now letís just leave it at that. So DNA polymorphisms, thatís where the action
is going to be when weíre talking about DNA profiling. There are many places where DNA between two
individuals is likely to be the same; that might be important for giving us eyes and
hair and fingers and so forth, but itís not going to be useful for distinguishing between
people. We want to look at the spots that are likely
to be different, the DNA polymorphisms. Hereís another word thatís going to come
up frequently when weíre talking about DNA testing and DNA testing results, this idea
of a locus or in plural itís a Latin word, loci. A locus is simply a specific location on a
chromosome. In fact the root words are the same right,
location, locus ñ thereís some commonality in the sounding there because they have the
same Latin root. All weíre talking about is a specific spot
that weíre paying attention to along the length of an individualís DNA molecules. And one last vocabulary word for this slide,
when weíre talking about a polymorphic region, that locus that has polymorphisms, what weíre
talking about is a region where we can distinguish between one version of the DNA we might find
and another version. In those circumstances weíre talking about
two or more alleles; an allele is simply a recognizable variant of a DNA molecule that
you might find at a particular locus. So again, I apologize for the vocabulary,
but it will make things easier down the line if weíre all using the same words to mean
the same things while weíre talking about DNA profiling, and these three come up time
and again. There are two others I just want to go over
quickly right now, and that is this idea of PCR; Iíve already mentioned PCR a little
bit earlier in this particular video. Youíll find that molecular biologists like
to use abbreviations. It all starts with DNA, an abbreviation for
deoxyribonucleic acid. I already mentioned to you RFLP; well hereís
another one that molecular biologists are fond of ñ we like abbreviating things like
this, giving them fancy names and then breaking it down. PCR is short for the polymerase chain reaction. And the long and the short of it, PCR is a
very clever trick that molecular biologists can use to amplify DNA in their laboratories. Not just any DNA either, but specific regions
of DNA that we can choose which ones weíre amplifying by deciding what kind of primers
we add to the mix. In very simple terms, many people refer to
PCR as molecular Xeroxing. In the same sense that you can use a copy
machine to make many copies of a particular piece of paper, you can use PCR to make many
copies of a particular fragment of DNA. Now hereís one last fancy sounding word ñ
electrophoresis. The bottom line for electrophoresis is this,
itís the way that weíre going to separate DNA molecules on the basis of their size. The ìelectroî means weíre going to use
electricity, the ìphoresisî is talking about movement; weíre going to pull DNA molecules
along. Big molecules will have a hard time moving,
small molecules will zip along. We can separate DNA molecules on the basis
of their size using this technique of electrophoresis. But enough vocabulary, letís get to the topic
at hand; letís get to work talking about how it is that automated STR testing actually
works. So, hereís the name of the game. Ultimately when weíre doing DNA testing,
be it automated STR tests or any of its preceding varieties, the objective is to get DNA, some
genetic material from a reference sample, here shown as a vial of blood, and compare
it to that same genetic material that we get from an evidence sample, here a bloody handprint. And so the first step in this process is quite
simply to extract and purify DNA from both of these two different kinds of material. Iím not going to get into the details in
this relatively short video about how it is that thatís accomplished, but I hope youíll
take my word when I tell you that itís actually a very simple, straightforward procedure. Itís rooted in the very same principle for
getting stains out of laundry. Iím not exaggerating, thereís no hyperbole
there. The way that DNA might be lifted or taken
out of a bloody handprint is exactly the same principle ñ warm, soapy water, a little bit
of agitation, put a little bit of salt in to move things along is the first step in
actually getting DNA and cellular materials off of a substrate that it might be associated
with. Itís the first step in an extraction. The purification steps are not that much more
complicated. There are kits that can help move that along
very quickly, but you can do it old school and just use a couple of chemicals. Phenol chloroform is one, and then follow
that with an ethanol precipitation. It sounds fancy but in practice first graders
can do these extractions and purifications easily without any risks to themselves using
just a couple of pennies worth or reagents in the span of just a few minutes. Extraction and purification of DNA is nothing
that anybody should be afraid of. Thereís one thing to bear in mind when weíre
talking about extracting and purifying DNA, and thatís this ñ it turns out that sperm
cells are a little bit different than all of the rest of the cells in our body. They have tough protein coats that allow them
to stay behind in certain solutions, whereas other skin cells, or other cells like skin
cells might have gotten broken up. That in turn allows this idea of a differential
extraction. In sexual assault cases youíll sometimes
find that a testing laboratory has taken some pains to try to separate the sperm cells from
the other cells in a particular fraction. When that works it can give us a nice clean
look at the contributor of male DNA to a particularly mixed sample, but when it fails weíre typically
in no worse shape them we would have been if there hadnít been an attempt to do that
differential extraction. So there is some slight variation you can
do with the extraction techniques but for the most part most extractions are done the
same for al samples regardless of the crime or their likely sources. Alright, where are those extractions and purifications
going to take place? The short answer to that particular question
is in one of these kinds of test tubes that you see here on this particular slide. They have a fancy name, theyíre called eppendorf
tubes, sometimes called microfuge tubes. But this slide doesnít do justice to just
what it is weíre talking about here with an eppendorf tube. Let me show you an actual eppendorf tube here
in my hand. This particular eppendorf tube is one thatís
designed to hold 200 microliters within it, so a fifth of a milliliter, much more than
you would typically use for a typical DNA profiling experiment. So this is the kind of test tube. I know many of you watching these videos have
probably taken high school chemistry labs and such. The test tubes that you worked with in that
setting are very different than the kind of test tubes that a molecular biologist will
work with. And so you can see the test tube here has
a little lid thatís attached to it and we can seal it and reopen it and put things in
and take things out of such a tube during the course of doing a DNA extraction and purification. Now, what is it you use to move materials
around to and from a tube like this? The answer to that question is a fancy instrument
that we call a pipettor. While weíre on this slide let me point out
that weíre talking here ultimately for a typical DNA test in the ballpark of 10 to
20 microliters is the final volume of liquid. And remember, a microliter is a millionth
of a liter; a microliter is barely visible to the naked eye. 10 microliters you can see unless youíve
got really bad eyesight, but itís still a really small amount of material. And itís important to bear in mind that DNA
doesnít have any special color associated with it. Weíre often taking, as a matter of faith,
that weíve succeeded in moving DNA from one tube to another or that a particular reaction
is working the way we had intended it to be working. Itís often only at the very end of the process
that we know if things have worked out quite right. So I have here with me an actual pipettor,
just like the one pictured on that previous slide, and I think itís worth pointing out
that this is a fairly easy to handle instrument; youíll find these literally in every molecular
biology laboratory. You canít do molecular biology, let alone
DNA testing without one. And thereís really not that much to the instrument. Thereís a plunger up here at the top that
you can press down upon. And if you look really closely you can see
my thumb is just barely moving, thatís enough pressure to cause the tip of the tube now
to be ready to accept one microliter or liquid from a tube like this one. So when I lift up on the plunger the tip of
the pipettor now has within it one microliter of liquid. And so thatís how it is that we would move
around such a tiny amount of material. Itís obviously important to calibrate an
instrument like this; if youíre off by a factor of two or three when youíre dealing
with only 10 or 20 microliter of total volumes that can obviously have some important consequences. And one other feature of a pipettor thatís
going to be helpful to bear in mind if you have concerns about contamination and things
of that nature is this tip; the very tip of the pipettor is actually a disposable piece
of plastic and it can come right off. So in one hand Iíve got the tip, the other
hand Iíve got the pipettor and I can always use this pipettor again now without worrying
about cross-contamination by getting another tip, such that we can do the whole process
one more time. And the tip can come off and is disposed of
after each use. So, that is the kind of instrument that weíre
going to use to move around these small volumes of liquid. Letís talk now about the machines that are
used in the lab to do the PCR amplification process itself. Here on this slide is an image of a thermal-cycler;
thatís the name of the particular instrument that would be used in a laboratory interested
in doing PCR amplification. Let me tell you that an instrument like that
isnít that large; itís about the size of your typical breadbox. Theyíre very widely available. Theyíve been used in molecular biology laboratories
for going on 20 years now. You can purchase used ones of these for a
few hundred dollars off of the internet, if you were so inclined to do that. They donít take up a lot of space and theyíre
not that expensive. The bottom line is this instrument does a
good job of cycling a sample, the eppendorf tube through three different temperatures. And that in turn is what drives the PCR process. This next slide actually gives you a little
bit of a closer look at a molecular level to what it is thatís going on during the
course of PCR. As I said before, PCR is essentially a molecular
Xeroxing machine. Whatís going to be happening is that with
each round of PCR you will double the quantity of a particular region that you might be interested
in. This slide actually shows you some of the
specifics of how it is that that plays out, but for the purposes of this video, let me
simply say that with each round of PCR amplification we double the amount of material that we were
starting with that we might have been interested in. And if you look at this part of the slide,
youíll see that for a typical forensic investigation, the test kits that are used recommend that
there be 28 rounds of amplification. You know what that translates to? 28 rounds of doubling of a region of interest;
quite simply we go from talking about looking at a particular region of DNA thatís like
a needle in a haystack to at the end of 28 rounds of amplification, two to the 28th times
copies of that particular region, to finding ourselves to having a stack of needles with
maybe a piece of hay hidden somewhere within it. Thatís the advantage, thatís the power that
comes from PCR amplification. Along the way, as part of the PCR amplification
trick, itís also possible to fluorescently label the fragments so that they glow in different
wavelengths of light when we shine a laser beam at them, thatís going to come in handy
in terms of tracking where these fragments have gone in the next step of the process. And speaking of the next step in the process,
for that weíre going to need another instrument still ñ this is the one that might break
your bank if youíre thinking about setting up a crime laboratory in your own garage or
basement. This here is a picture of an ABI 310 Genetic
Analyzer. There are other variants of this machine that
are now available ñ a 3100, a 3130 ñ but the basic principle is all the same, and this
particular instrument is still the workhorse for most crime laboratories. The principle is pretty straightforward. The business part of the instrument is going
to be right here. If you look very closely you might be able
to see a very faint gray line. That is the capillary in which capillary electrophoresis
will occur. Donít worry; the next couple of slides will
help you get a better understanding about whatís going on there. But again, the most important part of the
slide is right there. This next slide shows us a cartoon that comes
from the userís manual for an ABI 310 Genetic Analyzer that lets you see things a little
bit more clearly. When I, as a molecular biologist, talk about
DNA, the very first thing that actually comes to mind to me is that itís an intensely,
negatively charged molecule, and I think we all appreciate that opposites attract. So what we can do is, if we start our DNA
off at one end of the capillary and put an electric charge down there by applying an
electric field, what we can do is if we start our DNA over here at this end of the capillary
itís naturally going to be drawn to the positive electrode; itís going to go with that electric
current. And thatís exactly whatís happening during
capillary electrophoresis. So, DNA is going to get pulled through this
very thin, hollow tube; from negative electrode to positive electrode. And along the way, big molecules of DNA are
going to have a hard time fighting their way through that little narrow tube, in the matrix
thatís within it. Medium sized ones will get through a little
bit more easily, and the ones that move through the quickest of all will be the smallest fragments
of DNA that there are in a particular sample. Eventually though, regardless of their size,
small, medium or large, theyíre all going to parade past this one part of the instrument
thatís called the detector window. And the detector window is very simply a CCD
camera, probably much like the one that you have in your cell phone, paired up on the
other side of the capillary with a laser. And as these DNA fragments are moving past
the laser, they get excited; they fluoresce in a different color because of the labels
that got put on during the PCR amplification. And those colors get picked up by the camera. And so again, eventually all the DNA moves
past that detector window, and all of that information gets transmitted to a computer. And this is what it is that that data looks
like as its being captured by a computer. Let me tell you what it is these axis mean. This vertical axis here is described in terms
of relative fluorescent units, or RFUs. Again, molecular biologists like their abbreviations. And all that weíre seeing here is a measure
of the intensity of light that that CD camera picked up upon at different points in time. And the horizontal axis here is measured in
terms of minutes, or seconds if you prefer. It starts at time zero and it proceeds all
the way through to about 30 minutes. From beginning to end it takes about 30 minutes
for even the largest fragments of DNA to run that gauntlet of the capillary in a capillary
electrophoresis machine. If you look at what weíre seeing with the
data here, youíll motive that not much at all happens for the first dozen minutes or
so for the whole electrophoresis process. Then we see a lot of light and a lot of different
colors. Each line here corresponds to a different
wavelength of light that the camera is detecting. These are small bits of DNA, theyíre actually
the primers left over from that PCR step earlier in the process. And after theyíve moved through then we start
to see this pattern of peaks, spikes and valleys essentially; thatís where the information
is, thatís where weíre going to be able to see some DNA profiles. So this information gets captured by a computer
and then computer software is used to tease apart these peaks into their component colors. Software also then is used to attach each
of these different fragments or peaks that youíre seeing along the way here. And the next couple of slides are intended
to help you get a feel for what it is these electropherograms mean. Each of these graphs corresponds to an electropherogram,
taken together, this is a DNA profile. And in fact, this is the DNA profile thatís
generated from the raw data that you saw on the previous slide. So what do we see when weíre looking at one
of those electropherograms? Well, let me tell you, hereís how we read
electropherograms. Those peaks that you saw on the electropherograms,
those each correspond to individual alleles. And thereís actually nine different loci
which weíre going to be getting information from that particular test kit that we used
on the previous slide. Each of those can have information, some peaks
that tell us what alleles are present there. And those end up showing up as peaks again
on the electropherogram. This is the same electropherogram that you
had seen in the previous slide, but letís take a closer look at the electropherogram. Itís easy to say that the computer has separated
the peaks into three different kinds of color. We have some peaks in blue, some in green,
some in yellow, shown in black because yellow doesnít print or show up well on computer
monitors, and also some in red. So four different color fluorescent dyes were
used to label the DNA in that PCR amplification, and within each of the three first colors
the blue, the green and the yellow, you can see there are actually clusters of peaks;
three clusters ñ three in blue, three in green, three in yellow. These are corresponding to the fragments of
DNA that have been amplified from specific loci that are part of this particular test. And each of those loci has a name, here are
their fancy names. The first one is often referred to as the
D3 locus; the second blue one is the vWA locus; the third one is the FGA locus and so on. But the names, while theyíre important and
they help us keep track of whatís going on, nobody expects an attorney to have committed
this sort of thing to memory. Hereís the thing that we think is interesting
when we look at these electropherograms ñ again, each of those peaks corresponds to
an allele that was present with the DNA sample that was being tested. And here is exactly what I mean by that ñ
here, if we have at the D3 locus, which actually has an even longer formal name, but if you
look at the D3 locus in this illustration you can see there are two alleles present;
one thatís a 16, one thatís a 17, thatís the name that we gave to it with the computer
software. And that the D3 locus on the electropherogram,
that in turn causes us to see two peaks, a 16 and a 17. And by the same token at the D13 locus, one
of the yellow loci, here you can see an 11 and a 13; that corresponds to alleles 11 and
13 that were present in the underlying DNA sample. There is one other locus that you may have
noticed, itís this one here in the green; the left most on the electropherogram. This is from a locus thatís not polymorphic,
so there are nine polymorphic loci for this particular test kit, one not so polymorphic
locus. The amelogenin locus is a gene that resides
on the X chromosome and the Y chromosome. The X chromosome version is a little smaller
than the Y chromosome version and the long and the short of it is this. If you find a DNA profile that has two peaks
at the amelogenin locus, guess what youíve found ñ youíve found a DNA profile that
came from an individual with both an X and a Y chromosome, in other words weíre talking
about a males DNA. In contrast, if you only see one peak, just
the X then that means that weíre talking about an individual who has no Y chromosome;
in other words, a female. And there are one last set of peaks to look
at here, these are the ones labeled in red. These arenít actually amplified during that
PCR amplification step; instead these are already pre-added to the mix. They are internal size standards. And itís by comparing where peaks move in
the yellow the green and the blue to where peaks moved in the blue that weíre able to
make size determinations for these samples. We know how big each of those fragments are. If a particular piece of DNA moves just a
little bit further than that 100 does that tells us itís probably an X, especially if
itís labeled in green. So thatís the sort of thing weíre looking
for in an electropherogram. Thereís more information that we can get
out of an electropherogram than just the DNA profile. Iíll be talking with you about some of these
specifics in greater detail with some other videos, but for now, letís talk about some
of the basics. Remember weíre looking at nine different
polymorphic loci plus the amelogenin locus, here is a quick insight you can get just at
a glance. If at a locus you see two peaks, what that
means is that weíre talking about a locus that we would call heterozygous; very simple
direct translation ñ if there are two alleles, two peaks that means weíre talking about
a heterozygote individual at that locus. And thatís in contrast to what you might
see at a locus where thereís just one peak. In that circumstance we call that a homozygous
locus where the individual is a homozygote. If ever you find a locus that has more than
two peaks, in other words three or more, the simplest interpretation is that weíre talking
about a mixture of DNA from more than one individual. The bottom line is human beings, weíre diploid. We have two copies of our genetic instructions. Sometimes we get the same instructions from
both our mother and father; that makes us a homozygote. Sometimes we get something from mom then what
we got from dad; that makes us a heterozygote at that locus. Itís very unusual to find that we had information
thatís more than just from mom and dad. There are some known instances but those are
rare. So those boxes are showing you a heterozygous
and a homozygous locus respectively. Now the position of peaks also tells us something. The peaks that are on the left side of a set
of electropherograms correspond to smaller fragments of DNA. The ones that are on the right side correspond
to larger fragments. Because remember, whatís happening here is
all of these peaks correspond to DNA thatís being pulled through one of those capillary
tubes during capillary electrophoresis. The first things that get recorded show up
on the left. That corresponds to a smaller set of DNA fragments
then these; they come up further on the right. So if youíre looking at an electropherogram
size is always described in terms of small on the left, large on the right. And the heights of the peaks is something
that can give us some interesting insights into a sample. Ultimately the height of a peak is proportional
to the amount of DNA that gave rise to that particular peak in the PCR amplification. So let me draw your attention here to this
locus; this is a homozygote. The individual contributed two copies of the
16 allele. Over here they contributed one copy of the
14 and one of the 15, notice the difference in the heights of the peaks. The homozygote locus is twice as tall as the
heights of the peaks at the heterozygous locus. So letís move on. Here in a nutshell are all the peaks that
you might see with a DNA profile test, like Profiler Plus. This is just showing you all the most commonly
observed alleles so you can see thereís quite a few that you might be able to find for any
given sample. Again, for an unmixed sample you would expect
to find at most just one or two, but these are all the ones that you might find with
the Profiler Plus test kit, one thatís been very commonly used in crime laboratories over
the years. Hereís the kind of peaks that you might see
if you used a different test kit; this is called SGM+. Itís from the same manufacturer but this
particular kit is more popular in Europe and especially in the United Kingdom. But ultimately again, this is the kind of
DNA profile that youíll see when you are generating a DNA profile with the Profiler
Plus test kit. And it brings us then to asking questions
like ìhow impressed should we be if we find that the DNA profile for an evidence sample
is the same as the DNA profile that you found in a reference sample.î In other words, what do we mean, how impressed
should we be if we find that two samples match. What is the weight that should be given to
a DNA profile match? To answer that question we need to actually
consider three alternative hypothesis. The first hypothesis is the one that the prosecution
always gravitates to, and that is simply this, the reason that the DNA profile between an
evidence sample and a reference sample match is because they have the same source. Thatís the power of DNA profiling. But there are two alternative hypothesis that
need to be considered as well. One is maybe itís just the result of coincidence;
maybe the perpetrator of the crime just by chance happens to have the same DNA profile
as some hapless suspect who has been incorrectly charged with a particular crime. I said at the very beginning here for this
video that the chances of that happening for a good clean unmixed sample with these automated
STR results is vanishingly small. These days itís described in terms of quintillions
or quadrillions. So the chance of a coincidental match in many
instances can pretty effectively be ruled out of consideration. But Iíve said thereís a third possibility,
and that third possibility is that maybe thereís been some sort of a mistake in the process. Maybe there was a mistake in the collection
of the evidence. Maybe thereís a mistake in the handling after
the collection. Maybe thereís been a mistake in the manipulations
within the laboratory such that thereís been some type of contamination. Maybe there was a mistake that was an accident. Maybe there was a deliberate mistake. Al of these things are possibilities and all
of those things need to be considered when weíre deciding just how impressed we are
with a DNA profile match. Thatís more than I think we want to get into
for this particular video, but instead itís something that weíll be talking about in
subsequent videos in this series. Soon Iíll tell you all about how it is that
we would generate a statistic for a straightforward sample, and then not long after that weíll
talk about how it is we generate a statistic for a more complicated sample where a mixture
may have occurred or where there may be some other complicating factors. But for now I think we can wrap this particular
video up and simply thank you all for listening in and encourage you to come back so we can
talk some more in a little while about how it is we answer those questions about statistical
weights. Iím Dan Krane and I hope that youíve enjoyed
this video about how it is that DNA profiles are generated.

6 thoughts on “Generating forensic DNA profiles (HD version)

  1. A milli gram is 1/1,000 of a gram;A micro gram is 1/1,000,000 of a gram;A nano gram is 1/1,000,000,000 of a gram;A pico gram is 1/1,000,000,000,000 of a gram;

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