Hi. It's Mr. Andersen and in this podcast I'm going to talk about all of
molecular biology. Before I get there I want to talk about a story close to home. In the
1970s some researchers went to Yellowstone Park. They were in the geyser basin. They
pulled these bacteria out called thermus aquaticus and from that they pulled out an enzyme called
taq polymerase. That means thermos aquatic polymerase or the DNA polymerase found in
this bacteria. Cool thing about it is that it can handle really high temperatures and
survive. And the whole genetic engineering molecular biology is built upon this enzyme.
So we're talking about trillions of dollars. How much of that goes back into Yellowstone
Park? None. And so they've changed their policy on that. But it's a great way to kind of frame
this idea of molecular biology and how important it is. When I was thinking of how I wanted
to explain this in a way that makes sense I always kind of fall back on an analogy.
And so basically what I want to talk about is how we've gone over the last 30 or 40 years
to the point where we now know all the DNA inside a human. And so basically think about
it this way. Imagine if you look at these magazines right here there's a whole bunch
of information that's found inside here. There's a whole bunch of text inside here. And basically
if could I take all of the text that I want and make something out of it, like a really
cool ransom note, then I would be manipulating that text. And so I want to kind of use this
as an analogy for what we've done in molecular biology. And so what was the first thing that
was ever done? Well the first ransom note ever in the terms of molecular biology looked
something like this. And you might say that's not very impressive. Basically what is it?
It's like cutting out letters from a magazine but turning it upside down so you can't see
the letters. And so what was the first thing that we were able to do is we were able to
cut the DNA. And we do that using a restriction enzyme. But you should think of a restriction
enzyme like a scissors. It's able to cut the DNA. How do we paste DNA? Well basically to
paste DNA the glue stick equivalent of that is hydrogen bond. DNA will just come back
together again once it's been cut because of the hydrogen bonds. What's the next ransom
note? Well it would look something like this. Basically it was the first recombinant DNA.
We took DNA from a frog if I remember right. And a bacteria. And they were able to combine
those two using this hydrogen bonding. But again, a not very impressive ransom note.
Next thing that happened was something like this. So you can see in here that what we're
doing is actually ordering these little fragments cut out of a magazine from small to large.
But again you can't see what the letters are. So that's called gel electrophoresis. And
then we developed something called PCR, polymerase chain reaction, where we made a copy of that.
And so if this is that original, in PCR we make a duplicate of that. And we make an exact
duplicate. So every duplicate that we make from that is going to be exactly the same.
And that's what PCR does. It makes copies of DNA that's exactly the same. Okay. What's
the next advancement? Well the advent of the markers. So markers are basically going to
be a little section of DNA that marks that specific DNA. So if you've read that they've
found the marker or the genetic marker for breast cancer, that means that there is a
piece of DNA that's inherited by all people who have this genetically based breast cancer.
And then finally we get to where we are today. So right now we've used DNA sequencers to
figure out all of the letters inside our DNA. But it's hieroglyphics. In other words you
can't really read what it means. We know what all the letters are but we don't know where
the genes or what the genes express. In other words what proteins they make. And so when
we look at this ransom note, this is the future. This is where we want to get but we're still
quite a ways out. And so basically going back to the big things in this talk that you'll
need to remember. First one is the scissors. The role of the scissors will be played by
restriction enzymes. The roll of the glue will be hydrogen bonds that are found within
the DNA. The ruler or the measuring device is something called gel electrophoresis. To
copy that we use the polymerase chain reaction. And then to actually read it we use something
called a DNA sequencer or a gene sequencer. It's going to look at all of the letters even
though we might not know what they do, we can find them right now. So let's start with
the scissors. \b
\b0 And that's the restriction enzyme. Where do restriction enzymes come from? Well basically
they come from bacteria. Because bacteria have been locked in this million year war
with viruses. And the viruses you can see inject their DNA into the bacteria DNA. And
they make more viruses. And so how do you fight back? Well basically what they do is
they methylate their DNA. First of all what does methylate mean? You basically add a methyl
group to all of their DNA from the bacteria to protect it. And then they're going to secrete
these or create these restriction enzymes. And what the enzymes do is they chop up or
cut DNA. Since all of the bacterial DNA is protected by these methyl groups what it's
really going to chop up is all this foreign DNA. And so all of that viral DNA is broken
apart. And then the bacteria after it's done that can return to its specific shape. So
let me show you how restriction enzyme works. So if this is a section of DNA a real common
restriction enzyme is something called Eco R1. It come from the word E. coli. And this
is a restriction enzyme 1. And basically what it will do is it'll scan the DNA until it
finds this specific sequence and it's literally going to cut it in half. And so if I were
to find that, where is that going to be up here? Well you can see we have a G A A T.
So it's going to cut right like that through here. It's going to go all the way to the
end. And it's going to cut it in half like that. And so if we take a look at that basically
what will it do? It will break that into two fragments. If we take another one. Oh. Did
you see that? It came right back together again. So what brings it back together again?
There are going to be little hydrogen bonds here between those two end. And in fact we
call this a sticky end and this a stick end. Because there are going to be hydrogen bonds
that form between the two. And so once the enzyme's gone, it comes right back together
again. If we were to look at another one, this is the HindIII enzyme. Basically it's
going to cut between these two As. And so it would cut it right here. All the way down
here. And it's going to cut it into two fragments like that. If we apply that enzyme that was
found in this specific type of a bacteria, if it goes away then it comes right back together
again. What if we add both of those enzymes? What's it going to do? Well it's going to
cut it in two points or two restriction sites and then we're going to have 3 fragments that
come from that. So those are restriction enzymes. What do we use them for? In molecular biology
basically to cut DNA and then to glue it back together again. All you need is hydrogen bonds.
And so the first recombinant DNA, that of a frog and a bacteria, how did they get together?
Basically they cut them both with the same enzyme. They had the same sticky ends and
then they were able to come back together again. What's the next one that we have to
do? So what we've done is shown you how to cut. That's a restriction enzyme. How to glue.
That's hydrogen bond. Now we have to figure out how to separate the DNA according to its
length. And so to do that let me talk about Pachinko machines. Pachinko machines are essentially
these vertical machines that are really popular in Japan. You put a ball up at the top. You
usually flick the ball up at the top. And then that ball is going to bounce down. And
if it eventually goes where you want it to go, if it eventually goes into a little cup
down here at the bottom, then you might get more of those balls back. But in that case
you wouldn't. So imagine a game of Pachinko where we had one ball like this that's bouncing
its way down. And then we had instead of another ball we had like eight balls that are attached
together. So something like that. So what would happen to them if we dropped them in
a Pachinko machine? Does it make sense that they are sometimes going to go slower. They're
going to wrap around. It's going to take them way longer to get down to the bottom. And
so the single Pachinko ball would already be at the bottom where this big chain of Pachinko
balls hasn't even made it very far. And so how does that apply in DNA? Well think of
DNA instead of Pachinko. So basically if we were to take a small fragment of DNA it's
going to work it's way farther down through that Pachinko machine sooner than one of these
big fragments. And again we don't use Pachinko machines but we do you gel electrophoresis.
And so how does this work? Basically inside you have a gel. The gel is kind of the consistency
of jello. It's going to have little wells on one side where you insert the DNA. And
what's pulling the DNA? Well in a Pachinko machine it's going to be the gravity. But
here you can see that there is a red cable. That means there's a positive charge on this
end. So there's a positive electrode all the way across. There's going to be a negative
electrode. You can see it right back here on this side. So basically the DNA is going
to migrate across that gel. DNA has a negative charge which is going to be pulled towards
the positive end. And so if we look at this. This is kind of a, if you've ever heard of
like a DNA fingerprint. What's going on? Well basically the DNA is going to be pulled in
this direction. You could say this. Since it's been on for awhile that this fragment
right here is going to be bigger than this fragment right here. Because this small fragment
has made its way farther from these original wells where it was up here. And lots of times
you'll run a ladder as well. A ladder is a bunch of DNA with known distances or known
quantities. And so you can read across and see how big is that? How many nucleotide pairs
do we have? When we do this is class we use a dye called ethidium bromide. And basically
it will dye the DNA. You can put it under a black light and you can see where the DNA
is. It's normally clear. You can't see it. Okay. What's the next thing we have to do?
Remember we have to, now that we've sorted it according to its length now we have to
make copies of it. And to do that we use the polymerase chain reaction or PCR. This was
invented by Kary Mullis. He was riding his motorcycle one day and came up with this idea.
And so to do that we need a few extra things in our PCR machine. One thing we need is a
primer. Primer is going to be a little section of DNA that'll grab on to the DNA. It will
allow that polymerase to drive down the DNA. Now where is Taq Polymerase from? Remember
it's that bacteria. Because in the PCR we're going to heat this up really hot. What else
do we need? We need nucleotides. So we need new letters. And so let's check this out.
Basically we put it into a PCR machine which looks kind of like a photocopier. You put
your DNA in the top. And then it's going to quickly heat it and cool it and heat it and
cool it. And so as it heats this, it's going to unzip that DNA in the middle. It's going
to break all those hydrogen bonds. What's the next thing that's added? Well the next
thing that will be added is going to be the primer. The primer is going to bond to the
complimentary sides of the DNA. Now we create the primer because it's going to target a
specific gene that we want. What happens next? Well once the primer is in place, then taq
polymerase is going to grab on. Now this looks familiar. If you know anything about DNA replication,
how does that work? Well we unzip our DNA. It's helicase that does that. We put a primer
down. And then it's going to be DNA polymerase inside us that makes copies of our DNA. But
in a PCR machine it's taq polymerase. And the reason why is taq polymerase can withstand
these really hot temperatures. Okay. Watch carefully. What happens next? We basically
as the taq polymerase runs in either direction it's going to add complimentary letters to
either side. And so basically what do we have? We had one strand of DNA. Now we have two.
So the PCR machine will cycle. It will again heat up the DNA. So it unzips those hydrogen
bonds. We're going to add primer on either side. Taq polymerase is going to grab on.
And as it races down we're going to produce complimentary sides. And again. Heat it up.
We're going to unzip the sides. The primer is added on. Taq polymerase is on. And now
we have if you look at it we've now got eight strands of DNA. And so this is just a few
minutes we've gone from one to two to four to eight. And now we just get sixteen. You
get that exponential growth. So we can make a whole bunch of DNA very very quickly. So
we're almost to the end. What's the last thing in our analogy of the ransom note? It's really
reading what those letters are. And to do that we use a DNA sequencer. DNA sequencer,
sometimes it's hard to explain. Basically if you were to google the Sanger method, you'd
find some videos that might help a little more. But basically it's like a PCR machine.
So if you look down here we've got the primer, we've got the taq polymerase. We got out letters.
But then we have these four special letters. They're called dideoxynucleotides. And so
they're just like adenine, cytosine, thymine and guanine. But they have a specific color.
And so what happens you'll heat it up. Primer will be added. Taq polymerase will be added.
And then we're going to start adding those letters. So if I were to go across. If there's
a T here there's an A here. And a T and a G and a C and we'll say that's a T and a T
C G G C A A. Okay. So usually it's just going to make copies of it. But occasionally as
it makes those copies, instead of putting a regular A in, it will put one of these weird
As in there. What that weird A is going to do is it's going to be dyed. It's going to
have a specific color. And it's also going to stop the sequence. And so basically you
can't add new letters. And so a lot of the time you'll get a regular run of the mill
copied DNA strand. But occasionally you'll get these fragments that just have an A at
the front and they have a specific color which is going to be green. Maybe we run it again.
The next time the A seems normal. But the next one is a T that's weird. And so that's
going to stop it at that point but it's going to give it a color of red. Or maybe another
time we're going to get an A and a T and a G. But then we're going to put a weird C on
there. And that's going to stop it. And the C is going to have this blue color. And so
basically what you can do is then run gel electrophoresis. All these fragments are going
to run through a gel electrophoresis machine or run in a gel. And basically what they're
going to do is they're going to separate according to their fragment length. Well what's the
smallest fragment? Which went the farthest? That's going to be this little fragment of
A with the weird A at the beginning. And so what does that tell us? Well, it tells us
that the first letter is going to be an A. And so a computer can go through and read
all of these colors and it can read the sequence of the DNA. It can figure out the letters
in our DNA. And so the human genome project, that was the job of that. To sequence all
of the DNA in a human. These are the DNA sequencing machines. But what I want you to remember
is that we've sequenced all of the DNA in a typical human. And as you grow up they'll
be able to sequence all of the DNA specifically inside you. But at this point, we're kind
of right here. We've got the DNA? We know the order that it's in. But we don't know
what proteins those make or how those proteins interact. And so the next project after the
human genome project is the human proteome project. And so that's a lot. I know. But
I hope that's helpful.}