Professor Mark Saltzman:
So, we’re going to continue talking today about DNA.
In particular, we’re going to focus today on
sort of how to manipulate and use DNA in some applications,
and this is a huge area of science and technology.
You know this, you can – it’s hard to pick up
a newspaper or a news magazine without hearing some new
application of DNA technology. What I’m going to do is focus
on a couple basic things that turn out to be really important
for general applications, and then I’ll talk not in too
much detail about a few applications of DNA that you’re
probably familiar with to try to give you a framework to hang
this on. This, I think Chapter 3,
describes fairly well the things we’ll talk about today.
You’ve probably already noticed that there’s some material
that’s set aside from the text in boxes,
and I encourage you to particularly look at those boxes
for this chapter. There’s one on DNA
fingerprinting, for example,
that gives you a little bit more detail about that specific
technique; another one on production of
therapeutic proteins. I’ll talk about these both a
bit today but I encourage you to read those for more information.
I want to start where we left off last time.
We talked about the structure of DNA, how it works in terms of
a physical chemical model of the DNA molecules.
We talked about base pairing and how that leads to this
process of hybridization or very specific matching between
complimentary strands. We talked very superficially
about the biological process going from DNA to protein,
so the process of transcription,
RNA processing, and translation to produce a
protein. I ended with this picture that
shows you a little bit about control of gene expression.
The important concept is that, while every cell in your body
has the capability of making all the proteins that are needed
throughout your body, not every cell is doing that at
any given time. Only certain genes are being
expressed and it’s the family of genes that are being expressed
in a cell, likewise the family that are
not being expressed, that determines what a cell is
like, how it functions.
What’s called the phenotype of a cell and we’ll talk more about
this next week when we start talking about cells and a little
bit about cell physiology. There are multiple
mechanisms that a cell can use to decide which genes it is
expressing at any one time and which ones are not expressed.
I showed you this in this picture here and those levels of
control can be at the level of transcription.
There are molecules in cells that give the DNA the signal
that it’s time to transcribe and express a gene,
those are called transcription factors, we’ll talk about them a
bit later. There’s interfering with RNA
processing, and I’m going to talk about that in the next
couple of slides because there’s a couple of new methods for – or
potentially interfering with gene expression in living
animals that have been developed based on changing – interfering
with DNA transcription and the ability of messenger RNA to be
translated. You could interfere at these
later levels as well, for example,
by augmenting RNA degradation. If the messenger RNA for
protein is not present in a cell, then that can’t be
translated, obviously, and the protein can’t be made.
These two new medical therapies that I mentioned are
based on interfering with the biology of RNA,
and one is older than the other and the older one is called
anti-sense therapy. When you think about a gene or
a transcript, the messenger RNA copy of a
gene, you know that for every
sequence of a nucleic acid there’s a complimentary
sequence. Now of the two complimentary
sequences, one of them encodes the gene.
One of them has the right sequence of codons to specify
the amino acid sequence of the protein, and the other one has a
complimentary sequence. You know from our discussion
last time that these two complimentary strands are not
mirror images of one another, they’re not identical,
they’re complimentary. They face in the opposite
direction and you could predict the properties of one from base
pairing rules of the other but they’re not the same.
One of the strands encodes the protein, encodes for the
protein, the other does not. The one that does is called the
sense strand and the other one is called the anti-sense strand,
anti meaning it’s the compliment and it will hybridize
to the sense. What if you knew what the
sequence of a gene was? A gene, let’s say it’s the gene
for insulin. I’ll use that one as the
example because it’s a familiar one to most people and you know
that the protein insulin is made only in your pancreas and
certain cells of the pancreas. So that means those cells are
continually making messenger RNA and that messenger RNA is being
converted into protein. Well, what if you knew the
sequence for the messenger RNA that made insulin and you
designed another single stranded DNA or RNA molecule that was the
exact opposite, or the exact compliment,
I should say, of that strand?
So you made somehow an anti-sense polynucleotide to the
insulin gene or some fraction of the insulin gene.
Well, that anti-sense strand is shown here as the red
and the cell is naturally making the blue or sense strand of
messenger RNA for a particular protein.
If somehow you could take your anti-sense molecules that you’ve
made and you could get them into cells,
then by this process of hybridization they would
naturally form a pair like this. They would naturally hybridize
and form a duplex, or a double stranded nucleic
acid. When it’s double stranded this
gene can’t be translated, because you have to have the
single strand in order for the transfer RNA to bind and for
this process of translation to take place.
What you could do then, if you could deliver these red
colored molecules here is you could stop specifically the
expression of this particular gene in these particular cells.
Now, what are the challenges there?
You’ve got to be able to make this stuff and you’ve got to be
able to make it in large quantities and we’ll talk about
how to make nucleic acids in large quantities a little bit
later in the lecture. You’ve also got to get it into
the cell. It turns out that getting large
molecules like this, particularly large charged
molecules like nucleic acids, inside of cells is not so easy.
We’ll talk about that a little bit later as well.
In fact, we’ll talk about that concept throughout the course
because one of the big challenges of making these sort
of new biological therapies work in people to treat diseases is
getting the right molecules into the right cells,
at the right period of time. Now I gave you the example
of insulin and you probably wouldn’t want to stop insulin
production. That might not be a good thing
to do. What if this is a gene that’s
causing a cell to be cancerous? It was a gene that was causing
a cell to be malignant and to divide without control,
for example. Then you could imagine blocking
gene expression would be a therapy. Student:
[inaudible]Professor Mark Saltzman:
You wouldn’t want to stop insulin, for example.
In fact what you might prefer to do is start insulin
production and we’ll talk about ways to do that in just a
minute. These are ways to stop a gene
from being expressed, and there turns out there’s lot
of applications in that, lots of diseases result from
the unwanted expression of certain kinds of genes and
cancer is probably the best example of that,
but there are many. A newer version of this
that works in a similar way but a different way is called RNA
interference, and it turns out that this is a
natural mechanism that cells have.
It’s a mechanism that they have evolved in order to prevent
foreign genes from entering a cell and being expressed.
You have mechanisms inside your natural mechanisms inside your
cell that allow the cells to degrade unwanted RNA sequences.
Those mechanisms are called RNA interference.
You might have heard about this because it’s been quite an
active area of science. It turns out to activate
RNA interference, you deliver double stranded
RNA. Certain double stranded RNA
sequences will cause in the cell a process of degradation of very
specific RNA sequence. This involves mechanisms that
are still being understood, but if you’ve studied some
biology or read about this you’ve heard about the protein
complex called Dicer. Dicer is an internal cellular
mechanism for degrading RNA’s. You might have also heard about
the RISC complex, or the RNA silencing complex,
and these are the biological mechanisms that are involved
here and only shown by orange arrows on this slide.
The end result is you can design now very specific double
stranded RNA sequences, that when delivered into cells
again will activate this process of natural degradation of an
existing messenger RNA. Of course, if you degrade the
messenger RNA at a rapid rate than you’ll stop expression of
the cells. Now the nice thing about
this is that the degradation mechanisms seem to persist for
some period of time, beyond the time at which you
deliver the double stranded RNA; whereas, obviously,
this mechanism here is only going to exist for as long as
the anti-sense sequence is present.
So this might be a longer lasting, more permanent form of
elimination of expression of a particular gene. I just wanted to introduce
those concepts because you’ve read about them;
we’ll be talking more about RNA interference in particular as we
go on through the course. The rest of the time I want to
talk about expression of genes, of new genes.
Taking foreign genes, genes that aren’t naturally
expressed or might not even exist inside a cell and putting
them there and putting them there in a way where they work,
and by work meaning the gene gets expressed or translated
into a protein. I’m going to start by
talking about a very specific and interesting form of double
stranded DNA called a plasmid and plasmids occur in nature.
Plasmids turn out to be one of the most powerful and simplest
examples of a vector, what’s called a vector for
delivering DNA into a cell. Now the challenge is not just
to get the DNA that encodes a gene into a cell,
the challenge is to get it into the cell in a form where the
cell can use it, can express it and make
proteins from it. The plasmid has some features
which allow it to do that. Now to start with,
the plasmid is usually shown in a diagram like this as a circle.
It’s a double stranded circular piece of DNA,
meaning that the 3尧, 5尧
ends that hang off are joined back together again to form a
continuous loop. Again, these plasmids occur
naturally in nature; they were discovered
particularly in micro-organisms have plasmids that confer
biological properties onto them. This particular example of
a plasmid has several regions. Now, in your book,
there’s an example of plasmid where I’ve given you the exact
sequence of nucleotides that makes up the whole double
stranded DNA molecule. I just give you one of those,
right, because you could write down the other one because you
know the other complimentary sequence from base pairing?
One of the things about these plasmids that makes them very
useful is that their entire base pair sequences is known.
So you know everywhere on this picture you could write down
exactly what the sequence of nucleotides are that make up
this vector. This region here of this
particular plasmid is called the ori or origin of
replication. Remember we talked about how
DNA replicates itself and that there are enzymes,
DNA polymerase that bind to the double stranded DNA,
separate it, denature it locally,
and then start the process of replication.
One of the properties that you would like a plasmid to have is
you’d like for cells to be able to replicate it,
to make more copies of it. That way you could deliver a
small number of vectors and they could amplify into a large
number of vectors. So having a place that the cell
knows – where the cell knows how to replicate is important and so
plasmids have an origin of replication.
The blue region here is a gene.
It’s a gene that’s on the plasmid, and this particular
gene confers a specific biological property to cells
that have the plasmid and can use it properly.
The property it gives here is called AMP^(R) or resistance to
Ampicillin. Ampicillin is an antibiotic.
Antibiotics are chemicals, usually small organic molecules
that will kill micro-organisms like bacteria.
If a cell has a gene that makes it resistant to Ampicillin,
that means that that micro-organism can survive being
exposed to this normally deadly chemical without dying.
This is one of the biological properties, the naturally
occurring properties of plasmids,
is that they exist in microbial populations and they confer on
them resistance from toxins that would ordinarily kill them.
So being a micro-organism that got a plasmid that give you
resistance to an antibiotic would be a good thing – that
gave you resistance to something that naturally killed cells like
you in your environment would be a good thing.
We’re going to use that Ampicillin resistance in
technological ways and I’ll describe that in a minute.
The rest of this, this sort of beige part of the
molecule here is called the polylinker part.
This is where – this is the region of the plasmid where
we’re going to insert the DNA that we’re interested in.
We’re going to have DNA that we would like to make lots of
copies of, or we’re going to have DNA that we would like to
get expressed in a cell, and we’re going to put it in
this region that’s called the polylinker.
How we do that will be clear in a few minutes,
but this polylinker as is described down here is a site
where you can clone in genes. Let’s assume that we have
this plasmid cloning vector and we have some pieces of DNA that
we would like to put into a plasmid that we would like to
make copies of. DNA cloning,
or any kind of cloning just means ‘making copies of’.
So the process of cloning DNA is taking a few strands of DNA
of a gene that you’re interested in and making many copies of
them, that’s cloning,
you like to make identical copies.
This vector is going to allow us to do this.
The first step in the process is to take our plasmid
which we’ve selected, and to insert the gene that we
want into it. For a minute just assume that
we can do this and I’m going to show you how to do it on the
next slide. The first step is to take the
DNA fragments that we’re interested in and put them into
this vector by basically cutting open the double stranded DNA and
inserting the gene that we like in the region where we’ve cut.
Then we’re going to take the newly formed vectors that now
are recombinant, they’re combined from at least
two different sources. The sources are:
one, the plasmid vector that we’ve picked,
and the second is these genes that for some reason we’re
interested in. They might have come from two
completely different places, from two completely different
species from different parts of the world,
and they’re put together in a new way and that’s why it’s
called recombinant DNA. Then we’re going to take these
plasmid vectors and we’re going to somehow put them in contact
with cells in such a way that the cells ingest the DNA and
they use it. In this particular example
here we’re exposing these plasmids to bacterial cells.
That’s shown in this diagram as little colonies of bacteria that
are growing on a plate. You’ve probably seen agar
plates, if you smear a solution that’s contaminated with
bacteria on it, then that bacteria will grow on
this agar rich medium and you’ll get many, many copies of the
bacteria that you’ve smeared at low density onto the plate.
That’s a way of culturing or propagating bacteria.
Well, if you do that under the situation where you’ve put your
plasmid into these micro-organisms then you’re
going to have little colonies that grow many copies of the
bacterial cells. Hopefully each one of those
cells is containing one or more of the plasmids that you’re
interested in and those are being copied as well.
So what you get on the plate is many copies of the small number
of plasmids that you’ve put in. Now how do you find those
colonies on a plate that have the plasmid that you want?
Well, that’s a trick and there are multiple ways to do that.
One way is to allow these bacteria to grow on a plate that
is loaded with antibiotics like Ampicillin.
If this plate has Ampicillin in it, then the only cells that
would be able to grow here are cells that have resistance to
Ampicillin. If you selected the cells right
than the only ones that have that resistance to Ampicillin
are the ones that successfully got your plasmid and are using
this Ampicillin resistant gene. You could imagine strategies
where you have multiple resistance genes on a plasmid,
resistance to Ampicillin, to Penicillin,
to Erythromycin for example, and you design strategies for
separating out which cells are carrying the plasmid that you’re
interested in. This process of using a
biological event like resistance to Ampicillin in order to pick
out the cell population that you’re interested in is called
selection. If we grew these cells on a
plate loaded with Ampicillin and we could select cells that have
Ampicillin resistance, and this process of selection
and cell culture is very important and we’ll talk about
it more next week. How did we put our gene
fragments into this plasmid DNA in order to make multiple copies
of it, or to clone the gene? Well, it involves several steps.
The first step is we had to be able to take this circular DNA
and cut it to create a site for our new gene to be added.
That cutting is done by special proteins called restriction
enzymes. Restriction enzymes are just a
kind of enzyme, enzymes are protein molecules
that make a chemical reaction go faster,
and the chemical reaction that restriction enzymes do is
cutting DNA. They do that in a very special
way in that they – restriction enzymes are able to identify a
particular sequence of bases in a gene.
There’s whole families of restriction enzymes.
There are hundreds, thousands of them known now,
and each one has a specific character and one aspects of its
character is that it only binds and cuts at a particular
sequence of DNA. This particular restriction
enzyme here recognizes this sequence, GAATTC.
When it sees that sequence in a double stranded DNA it will bind
there and it will cut. Now another property of
restriction enzymes is that they always cut the DNA in the same
way. In this case,
this particular restriction enzyme cuts symmetrically like
this, but not at the same point. It doesn’t cut straight across
the double stranded DNA but it cuts in this jagged fashion.
That is, it cuts between the G and the A here,
and it cuts between the G and the A here.
When it cuts it leaves sticky ends or un-base paired single
stranded regions on each end of the part its cut and that’s just
a property of many restriction enzymes;
not all, some cut blunt, just right down the middle.
Most restriction enzyme also recognize symmetric sequences of
DNA, GAATTC for example. If you do the base pairing goes
exactly the same sequence backwards down here.
That’s an example of symmetric sequence and it happens that
most restriction enzymes also recognize those spaces.
If you cut and you open up a segment of DNA then you’ve
left these sticky ends, for example,
and these sticky ends are capable of recognizing each
other by the process of hybridization.
These will naturally want to reform and they’ll want to
reform to re-establish this base pairing.
They could be pasted back together, and the pasting
process takes advantage of this natural process of complimentary
hybridization. This gives you a biological
mechanism for cutting, using restriction enzymes,
and then you denature so that it falls apart,
and then you renature so that it comes back together.
Cutting involves enzymes called restriction endonucleases
or restriction enzymes, which I’ve already mentioned
and they have names. Restriction enzymes have names,
the particular one that does this function here is called
EcoRI. The names all look – they’re all italicized and
they’re capital letters and small letters so that they won’t
be easy for you to understand, but they are – if you know the
nomenclature, easy to understand.
This restriction enzyme was found in a natural source,
it was found in a micro-organism called E.
coli. The first three letters of E.
Coli are Eco, so Eco. It was found in strain R,
a particular strain of E. coli, and it was the first
one found, so EcoRI . There’s a nomenclature that’s
evolved for this. Now we know so much about
these, they’ve turned out to be so useful in biotechnology.
There are whole catalogs that you go to and buy restriction
enzymes. You can look up in the catalog.
What are the properties of this restriction enzymes?
What base sequence does it recognize?
How does it cut? What concentration do I need to
use to achieve that? So if you have plasmid where
you know all the base pairings than you could go through that
plasmid and say I want to cut it right here.
What restriction will do that? Or you could ask the question,
I have this restriction enzyme, at what regions on this plasmid
will it cut? The pasting back together
occurs partly naturally by this process of hybridization,
but hybridization only re-establishes the base pairing.
You know that these molecules are also linked in another way,
by the phosphate bonds that connect the 3尧
and the 5尧 carbons of adjacent nucleotides.
That doesn’t heal naturally but can be reformed by other enzymes
called ligases, ligases re-established the
phosphate bonds. How would I put a gene that
I’m interested in into a plasmid?
Well the first step would be to cut open the plasmid with a
particular restriction enzyme, and then what if I take that
same restriction enzyme and I cut up the DNA that I’m
interested in. If I cut both the plasmid and
my DNA of interest with the same restriction enzyme I’m going to
end up with the same sticky ends on both molecules.
Now if I put them in contact with one another,
the plasmid that’s been opened and fragments of the DNA –
special fragments that I’ve produced with the same
restriction enzyme, they’ll have the same sticky
ends, they will naturally hybridize with one another.
I apply ligase, and I’ve got the plasmid that I
had before but now with my gene, colored green here,
[inaudible]Professor Mark Saltzman: That’s a
really good question because if I open this up,
why wouldn’t it just reform with itself, why would it want
to have this in here? The answer is it will want to
reform with itself, and if I have these in solution
than how many reform with itself and how many reform with the
molecule I’m interested probably depends on the relative
concentrations of both in the solution and what conditions I
have it at. It’s a statistical process.
Some are going to reform and some are going to reform with
the gene in, and some probably aren’t going to reseal at all
under the conditions that I’ve used.
Not every plasmid in your test tube is going to have the right
gene inserted in the right way. One way that you can look
for that gene that you want is by making the cut in your
plasmid inside of a gene that encodes for some property like
resistance to an antibiotic. If this reforms,
so the plasmid reforms back to its native state,
that resistance will be recovered.
So bacteria that get an unloaded plasmid are going to
have resistance to antibiotics. If your gene goes in,
you’ve interrupted the gene for antibiotic resistance and those
new organisms aren’t going to be resistant to antibiotic anymore.
So you could use sort of negative selection in order to
find the ones that you want. I don’t know if that makes
sense or not but – Student:
[inaudible]Professor Mark Saltzman: Some do,
but there’s an advantage to having the sticky end there and
that you can put things back on, but there are also methods to
chemically produce a sticky end where you can take blunt ends
and you could add specific nucleotides onto one of the DNA
chains by either doing chemistry on a 3尧
or the 5尧 end and create your own sticky
end. Sometimes a blunt cut is useful
if you want to sort of grow a sticky end of your choice on it.
You’re starting to see that there’s all different ways that
one could take advantage of this fairly simple process of cutting
and pasting. That’s why molecular biology,
one of the reasons why it’s turned out to be such a powerful
tool, because if you can think
creatively you can find all different ways to using these
very simple principles to recombine molecules,
to make unique new DNA sequences. Where does the DNA sequence
come from? I want to spend a little time
talking about that. Say we’ve got a human gene that
we want to make and let’s say it’s the human gene for insulin
that we want to produce now. All the cells in your body have
the gene for insulin in them. Only cells in the pancreas,
some cells in the pancreas are making insulin.
One way I could try to find the gene for human insulin is to
take any cells from any of us, skin cells let’s say,
and I could identify where on the chromosomal DNA that insulin
is likely to occur. I could cut that up into
fragments, and I could search in these fragments to try to find
the one that has the insulin gene on it.
Now the problem with that is a problem I mentioned before,
that most human genes are not just a straight sequence from
beginning to end of the protein that you’re interested in.
There are encoding regions called exons and those are
interrupted by non-coding regions called introns.
If I cut up just DNA from the chromosome, what’s called
genomic DNA, then I’m going to have both exons and introns
within the fragments that I create.
That might be a good way to do it but it’s going to be more of
a challenge because you might – you’re going to have a lot of
these non-coding sequences that are in the way.
An alternate way is to go to the cell that’s making the
protein that you want. If it’s making the protein you
want, it must be producing messenger RNA with that gene one
it. That messenger RNA that’s being
used has already gone through the RNA splicing mechanism and
so the introns have already been removed.
If I could isolate that messenger RNA – messenger RNA is
just a copy of the DNA from which it came – so if I could do
the process of reverse transcription,
that is instead of transcription which goes from
DNA to RNA, if I could go backwards from RNA to DNA,
I could recover a DNA version of the gene that I’m interested
in. It turns out that we can do
that now because we have an enzyme called reverse
transcriptase, which is able to take single
stranded messenger RNA and make DNA out of it.
Now you’ve heard about reverse transcriptase someplace before,
right? Anybody heard of reverse
[inaudible]Professor Mark Saltzman:
HIV, HIV is a natural virus that contains an enzyme in
it. Why does it contain reverse
transcriptase in it? Because HIV is an RNA virus and
if it enters your cell the only way it can replicate,
it can put its DNA into your cells, is by first making DNA
out of its RNA genome. We’re going to talk more about
this later. Reverse transcriptase is a
naturally occurring protein, it has a biological function in
HIV, but we can use it for a
technical logical function here by going backwards on the
biological path from messenger RNA to DNA.
Now DNA that’s produced this way is not called genomic DNA
because this doesn’t match the DNA in your genome,
on your chromosomes, right?
The introns are gone now. It’s called cDNA or
complimentary DNA to indicate that it’s a copy,
a complimentary copy of the messenger RNA.
This is a much more efficient way to get DNA for a
gene that you’re interested for a couple of reasons.
One is the processing has already been done so the introns
are already out, so you don’t have to figure out
what’s exon and what’s the intron,
it’s already done for you. Plus, if you’re looking for
insulin, if you’re looking for the gene for insulin you’re
going to cells that are making it already,
they have abundant messenger RNA so it’s much easier to
separate out and identify the gene that you’re interested in.
You’re not fishing through a whole chromosome in order to
find what you want, but you’re going to a cell
that’s already enriched in it. Does that make sense? I want to talk about one other
technique and then I’m going to give you some examples about how
to use these in the last few minutes.
The technique I want to talk about is one called Polymerase
Chain Reaction. This is another way to clone or
make many copies of a gene of interest.
Now one of the big advantages of plasmids, I already
mentioned, is that you can take this plasmid and a plasmid is
one – you can think of it as a highly tuned machine for copying
itself. A plasmid is a highly tuned
machine for making copies of itself.
You take that plasmid; you put in the micro-organism
you’re going to make many, many copies of that plasmid.
We’re cloning a gene of interest to us by sort of
hijacking that biological mechanism by putting our gene on
this plasmid and so our gene gets copied many times,
as the plasmid gets copied many times.
That’s a way of cloning DNA. Here’s another – that’s a
biological way of cloning it. We’re using biological
mechanisms and we’re growing cells in order to accomplish it.
Here’s a way that you can do it in a test tube without
using any cells by this process called Polymerase Chain
Reaction. I’ll describe how it works here.
You have a fragment of some kind of DNA that you would like
to clone or make many copies of. Now in general these are small
fragments. They’re not whole genomes,
but they’re some small piece of DNA maybe the size of a protein.
You put this chromosomal DNA or this DNA that you’re interested
in, double stranded into a test tube.
You do that together with primers and with nucleotides,
because if you’re going to synthesize DNA you need the raw
material of DNA, you need the individual
nucleotides. You add a special DNA
polymerase called Taq polymerase is a polymerase that
was identified, a DNA polymerase that was
identified from an organism that lives in regions of the earth
that are constantly at high temperature.
Taq stands for thermos aquaticus,
it’s a marine micro-organism that lives near these
hydrothermal events in the bottom of the sea and they live
under very high temperatures and pressures all the time.
So their enzymes are tuned, unlike our enzymes,
which are tuned for working most efficiently at 37°
centigrade, Taq is used to living at 90°
centigrade. So its enzymes operate most
efficiently at this elevated temperature.
You take advantage of that, you put it in a test tube,
together with nucleotides and primers and your DNA.
Now you start a process, a cyclic process,
where the first step in the process is denaturing the DNA.
You can do that by gently adding heat and making it basic,
but you make the two DNA strands separate.
When they separate, the primers that you’ve added
automatically bind through the process of hybridization,
and then you turn up the temperature to the optimum for
Taq polymerase and DNA synthesis starts.
The polymerase starts a process of replication of your DNA
sequences. You first separated your DNA,
let the primers bind, then turn on the enzyme,
and it makes copies of each one of these.
You then denature again, each one of these strands gets
separated, primers bind, turn on the polymerase,
a new strand is made. If you repeatedly go through
this cycle of denaturing, synthesizing,
denaturing, synthesizing you’ll get many, many copies of DNA.
The number of copies you get depends on how many times you do
the cycling. If you start with only one,
you have two pieces of DNA, then you’ll get 2 to the N^(th)
fragments after N cycles because each cycle you’re doubling the
number. This has turned out to be a
very powerful technique because it’s relatively rapid.
It’s totally synthetic, there’s no micro-organism
involved in making the DNA for you,.
You can do it very rapidly in a laboratory, and fairly
expensively now. It’s really been a powerful
tool in molecular biology because I might have a very
small copy of a gene that I’m interested in and I can make
enough copies that I can start to do something else with it.
You can also use PCR to identify specific genes that are
present in a biological sample. You can use PCR for
fingerprinting as well, for looking in some unknown
fluid for example, is a gene that I’m interested
in there? You could use PCR to do that
because if you amplify a gene in PCR, that gene had to be present
from the beginning. Well let’s talk about some
mechanisms for using this and I’ll start with a simple example
of how one can detect a gene in a fluid,
in a blood sample for example, where that gene is unknown.
This takes advantage of the very specific properties of
restriction enzymes. This particular example
involves the gene for sickle cell anemia.
Sickle cell anemia is a disease that affects a subset of the
population. It’s a single gene defect and
we know exactly where the genetic defect is in sickle
cell. It’s on the gene for hemoglobin
and the hemoglobin that you produce if you have sickle cell
anemia is not quite right because there’s one base
difference. If you have normal hemoglobin
you have this sequence here, CCTGAGGAG.
In sickle hemoglobin you have a sequence that’s only slightly
different within the gene, CCTGTGGAG,
so there’s a thymine that’s substituted for an alanine and
that results in making hemoglobin that’s not quite
right that doesn’t function in the same way as the normal
hemoglobin gene does. How could I identify in a
chromosomal DNA sample whether the sickle gene is present?
Well one way you could do it is by saying ‘if I have this one
base pair difference then this sequence is going to be cut by a
specific restriction enzyme that recognizes the sequence
CTGAGGA’. That restriction enzyme,
there is a restriction enzyme that does that and it’s MST-3.
If I took this same restriction enzyme and tried to cut the
chromosomal DNA of a sickle patient,
it wouldn’t cut at that point because the wrong sequence is
there. Here’s – how can I find that,
right? Here’s the difference between
normal hemoglobin and sickle hemoglobin, I can make – I can
see a chemical difference, I can exploit it,
how can I find it? Well the way to find it is
by using a process called electrophoresis and Southern
blotting. Electrophoresis is described in
the chapter here, I’ll just describe it briefly.
If you have DNA fragments, so this is DNA that you’ve cut
up into fragments using restriction enzymes for example.
You load them into the top of a gel and a gel is – in this case
it’s synthetic polymer gel but it looks very much like Jell-O.
I’m sure some of you have dealt with electrophoresis gels in the
past and it’s really just a slab of something like Jell-O where
you can put a sample at one end of the Jell-O,
your sample of fragmented DNA. Now you apply electrical charge
across that Jell-O. Because it’s a gel electricity
is going to move through the gel because there are ions in it the
same way – for the same reason you don’t drop an electrical
device into the bathtub because charge moves through water that
has ions in it. Charge is going to move
through this gel and DNA is charged.
It’s loaded with phosphates which are negatively charged.
They’re going to move from a negative pole to a positive
pole, so they’re going to move through this gel.
Well if you’ve created fragments here and they’re
fragments of different size, the small ones are going to
move faster than the big ones. So the DNA is going to get
spread out on this gel according to size, with the small ones
going farther and the large ones not going as far.
If I can run the gel for some period of time,
run the electrical field, spread it out.
Now if I can stain in some way, if I can somehow label the DNA
fragments that I’m interested in,
I could find out where those fragments are on this gel.
I would be able to predict their size, because how far they
moved depended on how big they were.
How would I label this DNA? The best way to label this DNA
is by designing probes or labels that hybridize with specific
sequences that you’re interested in.
This label is DNA that might be made radioactive or made
fluorescent, and it has a base pair sequence that is from some
other region of the gene that you’re interested in.
It’s only going to bind to fragments that contain that
piece of the gene, and it will make those visible
to you in some way so you can see where your gene traveled.
Now something similar to this is the basis of DNA
fingerprinting, that’s described in one of the
boxes in your book. In the specific example,
what are we going to do? We’re going to take a
chromosomal DNA, we’re going to digest it with
this restriction enzyme, we’re going to put it in this
tube and run it on a gel, and we’re going to see what
results down here. In a normal cell DNA that DNA
in the normal cell – this DNA gets cut by the restriction
enzyme, so the sickle gene ends up – so
that hemoglobin gene ends up in two pieces.
One that’s .2 kilobases long and one that’s 1.1 kilobases
long – kilobase is 1,000 bases. When I look for it here I’m
going to see two pieces, one that’s nearly the same
length and one that’s much shorter.
If this was a sickle patient, so they had this gene
instead it wouldn’t get cut and when I went to look for that
presence of that gene on this gel,
it would appear as one large segment instead of a large one
and a smaller one. The absence of this smaller
region tells you that this sample came from a sickle
patient. You can imagine other ways of
doing this, or ways of doing this same thing in different
ways. It takes advantage of the
specificity of the restriction enzymes, the fact that we know
what the gene sequence that we’re looking for,
and using this technological process of electrophoresis to
identify changes that we predict. Student:
[inaudible]Professor Mark Saltzman: Yeah,
this is just one particular example of how to do it,
but you could – so you have to identify something that’s unique
about it and then design a method for identifying that
unique thing. Here the unique thing was that
there’s a restriction site inside that is present in normal
DNA and not present in sickle DNA.
If you could identify some other change you could do it as
well. Another example of using
this technique is to produce therapeutic proteins from cloned
DNA and I’m going to describe this one.
It’ll probably be the last example I have time for and so
I’ll go through the rest of them quickly in the next lecture.
But here, for example, the idea is to make many copies
of a protein for use as a pharmaceutical.
Here’s an example where we’d like to make insulin,
or having the insulin gene would be useful,
but if we could take the insulin gene and make many
copies of the insulin protein that would be a very useful
thing. It turns out that there’s lots
of proteins that have value as therapeutics and there’s a list
of them here and some of them you’ll recognize.
Erythropoietin, commonly called Epo and its
function is to treat anemia because it stimulates blood cell
production. You might think that its
function is to make you more – a better cyclist but that’s not
its only function. It’s used to treat people that
have – you’ve heard about Epo and blood doping, but it’s a therapeutic protein
as well and very useful for patients with anemia.
How do you make many copies of cloned DNA?
Well one way is to do just exactly what we talked about
before. Take a plasmid,
cut it open, insert a gene that we want into
the plasmid, and then put that plasmid in a host cell,
and let the host replicate it. Now in this case the host – you
want the host not only to replicate all the DNA,
you want it also to express the gene.
You’re not just trying to clone the DNA, you’re trying to also
clone or make many copies of the protein.
So that’s a slightly different thing, right?
We want not only for the cell to be able to synthesize the
DNA, you want it to be able to express the protein off the DNA
as well. That requires design at a
different level, not only does it have to have
an origin of replication which works so that you can synthesize
the DNA, the gene has to be inserted
together with a promoter and that promoter is a sequence,
a DNA sequence that the host will recognize as a signal to
transcribe and translate this protein.
We’ll talk more about promoters as they go on,
for now let’s just assume that we have a promoter that works in
this particular cell. The promoter in this case is
called the lac promoter and normally in micro-organisms
that lac promoter is used to produce a gene called
lacZ which makes a protein called
beta-galactosidase. What we’re going to do is take
this plasmid, with its promoter gene
construct in it and engineer it, to take out the lacZ
gene, leaving in the promoter and putting in the protein that
we want – the gene for the protein that we want.
In this case, it says it’s GCSF,
but it could also be insulin, for example.
Now when we put insulin in this place behind the right
promoter, the cell thinks that it still has the lacZ
gene present which it needs for its metabolism.
So it’s going to express this gene, but you’ve put a foreign
gene in the place of the natural gene, and so the foreign gene
gets expressed instead. In this case,
GCSF, or it could be insulin and now if I take this cell and
I grow it under the right conditions,
that is, under conditions in which the cell would normally
express the lacZ gene, it’s going to express our gene
instead and presumably, hopefully, make large
quantities of it. This was a process that was
perfected on an industrial scale by the company Genentech in
California, in the 1970s. The first therapeutic protein
that was produced was insulin to treat diabetes.
Before that, diabetics had been treated with
insulin that came from a different source,
usually from pigs, so by harvesting and purifying
insulin from pig pancreas. Now most diabetics take human
insulin made, not in humans,
but made in micro-organisms that are growing in a
manufacturing facility. That is a way of putting a
cloned gene into a micro-organism and than using
that micro-organism as sort of a factory for the protein.
You could do the same thing in mammals and here’s one quick
example of that, using a different kind of a
vector, but inserting a gene into the fertilized egg of a
mammal, in this case, a sheep.
The biology of this is more complicated than what I
described before, but you can inject this DNA
into a fertilized egg of a sheep and then implant that fertilized
egg with the foreign DNA that’s been micro-injected into it,
into a foster mother. If that leads to the birth of a
young sheep, hopefully that sheep has this gene encoded in
its genome now, and it will express the protein
that you wanted. One example of a way people
have used this, is that they’ve taken the gene
that ordinarily produces a milk protein.
They put a gene like the insulin gene in place of the
milk protein, but behind the promoter that is
used to produce milk proteins. So this gene is going to be
turned on in the sheep when it makes milk, under conditions
where it makes milk. If the sheep grows up in the
right way, and people have shown that this will work,
you can milk the sheep, the sheep has milk,
the milk has insulin in it. Here’s an example of using a
large animal by inserting the gene that you’re interested in,
you can make this animal into a factory for the kind of protein
that you would like to make. I’ll finish talking about these
examples at the beginning of lecture on Tuesday.
I’ll see you this afternoon in section.