Molecular Biology

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.}


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