WEBVTT

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What we've talked about in
recombinant DNA so far is how to get

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a piece of DNA from somewhere and
make a whole lot of copies of it.

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So, instead of working with DNA
extracted from my cells,

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which there's 3 billion different
base pairs of sequence,

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we can find a little stretch of DNA.
But simply being able to clone it,

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it was a long way from him being
able to get a lot of a fragment of

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DNA to be able to figure out what
that sequence is.

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That sequence of that gene is
actually from the [zero derma?

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gene, the gene that's broken and
the zero derma pigmentosa variant

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patience, missing one of these
translesion DNA polymerases that can

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copy over a thymine,
thymine, [permadine?],

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[dymer?], this induced by UV light.
So the reason they have that

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problem with their skin after
sunlight is because they are missing

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a polymerase that can't copy over
accurately over this very common

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lesion caused by DNA damage.
But how do you get from having a

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piece of DNA and having the sequence?
So, the first thing that people

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learn to do, and you still do this
all the time in any molecular

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biology lab.  We're sort of
switching now in engineering as

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you'll see.  You're going to see in
the next things that I'm going to

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say, proteins that were,
I talked to you about because of

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their biological roles.
DNA polymerase is,

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ligases, we learned what they do.
And now you're going to see them

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used in manipulative ways.
Restriction enzymes: they have a

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biological purpose,
too.  They weren't put on Earth for

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me to cut up into fragments and
clone.  They were there to give the

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bacteria is some kind of primitive
immune system.

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But the first thing that you often
have to do, and you have,

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let's say, a plasmid into which
we've inserted a fragment.

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And let's say it was the kind of
cloning we described the other day,

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where I had cut to the vector with
an ECO-R1 site,

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and the other DNA with an ECO-R1
site.  So these,

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the junction between the inserted
fragment and the cut vector has

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re-created two ECO-R1 sites now.
And if I cut with ECO-R1, I'll just

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undo what I did in the cloning,
and we should get the vector DNA and

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the insert DNA back again.
So, I can go from this vector to

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give us an orientation.
I'm going to imagine that it has

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one more restriction site.
This one is called [sal 1?

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.  It just recognizes a different
sequence.  So,

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if I take the plasmid DNA and cut
with ECO-R1, I just reversing

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the cloning.
So I should get is the vector DNA

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and the insert that I generated in
the first place.

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But I have to detect them somehow.
Unfortunately, they don't just look

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in the test tube like that.
So, what people do is they use a

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very simple principle.
It's called gel electrophoresis.

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And the idea is you just make a gel

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of something.  In this particular
case, it's just made of augur,

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which is agarose.  These are
polysaccharide products,

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often derived from seaweed or
something like that.

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They have the property that if you
warm them up, they're liquid,

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and that if you let them cool down,
they're a gel.  You've run into

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Jell-O which has the property.
That's actually made of a protein

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rather than a carbohydrate.
But it's that kind of principle.

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So it's very easy to pour something
and that let it solidify.

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Now you've got a slab.  And it's
just a network of things that

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interact.  And the principle of the
thing is that,

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so you have to get the molecules to
move.  Well that's pretty easy with

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nucleic acids because
they're charged.

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They have all those phosphates.
They've got a lot of negative

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charge.  So if you apply an electric
field, they'll move.

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And the principle of the thing is
that if you're big,

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it's harder to wiggle through this
network than if you are small.

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Or you can think of a big, fat
person trying to go through a forest

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with a lot of trees,
and a little skinny one.

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And if we let them have a race,
eventually the skinny one will

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emerge from the forest first.
And so, if we had a set of markers

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down on the side where this is big,
and this is small, and we take this

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piece of DNA, we're going to get two
fragments.  The bigger one would be

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the vector and the smaller one would
be an insert.  So from that,

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we could say, oh, if I didn't know
what I started with I could say what

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must be at that plasmid?
Is the vector?

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And I can run at all by itself and
see it's exactly the same size.

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And I got an insert of this
particular size.

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And now, can I learn anything more
about that just using restriction

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enzymes?  And let's say
I now take the same.

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Actually, maybe I'll do it over here.
So let's cut,

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this time, with ECO-R1 plus another
restriction enzyme.

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They all have these weird names:
[bam H1?], and let's see what

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happens.  Well suppose I do that and
I get something like this.

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Well it looks like the vector wasn't
cut at all.  That still seems to be

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the same, but it looks as though the
insert got cut into two pieces.

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Since it was linear, it must have
one site in it.

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And so, this molecule that I cloned
could look, be one of

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two kinds of ways.
It could be like this.

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Let's say this is the insert.
Here's the ECO-R1.  I'll use this

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sal 1 to orient us.
So, the bam site could either be

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close over here.
Or it could be over on the other

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side.  Does that make sense?
The logic is pretty simple.  How

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can I tell which of those is correct?
Just doing the kind

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of stuff I'm doing.
Beautiful, beautiful.

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So if we cut with the sal 1 plus
the bam H1.  In one case,

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one would get a fragment like that.
In the other case, get a fragment

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like that.
That should feel uneasily familiar

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to you.  It should feel just like
what we are doing what we did that

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phage cross, and we had some genes
that were lined up.

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And we were trying to figure,
was the orientation this way?  Or

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was the orientation that?
That was exactly the same principle.

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And so this is usually,
in the lab you'd call this

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restriction mapping,
or making a restriction map.

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And it enabled people to manipulate
fragments of DNA and make inferences

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about their orientation and other
features before we can actually even

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sequence DNA.
And that's just part of routine sort

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of stuff you do a lab.
The equipment is disarmingly simple.

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It looks something like that,
usually you're putting some colored

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dye so you can see that the things
are moving down the gel.

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And the way you visualize the DNA
is you add a molecule.

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The name of it doesn't particularly
matter.  It's called ethidium

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bromide.  But its property is it
doesn't fluoresce when it's just in

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solution.  But it's a flat molecule,
and it can interpolate in between

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the base pairs in DNA.
They have all those stacked base

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pairs going down a helix.
This molecule's flat, edit likes to

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slip inside.  And now it's a much
more hydrophobic environment.

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It's hidden from the water, becomes
florescent.  And so,

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DNA that's soaked up this dye then
will fluoresce when you put a UV

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light on it.  So if I take the gel
out of there after I've run it,

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and soak it in this dye and then
shine a little handheld UV light on

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it, it would look something like
that if I photographed it.

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And so, you would end up with those
patterns that look exactly like that.

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Oops, I guess I took the other one
out.  But you can,

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of course, depending on how
complicated it is,

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you could have a lot of different
fragments.  OK,

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so the next big thing that had to
happen in order for us to really

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move to where we are in today's
molecular biology was somehow,

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DNA had to be sequenced.
And as I say, when I was an

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undergrad, or even when I was just
about to start,

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when I was a postdoc anyway,
just again it seemed like how would

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you ever do it?
Because every nucleotide was joint

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by a phosphodiester bond.
The only difference was the base

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that was there.
It seemed very, very difficult.

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It was hard to imagine you would
ever be able to sort out the

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sequence of a billion base pairs.
Of course, you could clone.  Now

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you've got maybe a fragment of DNA
that's a couple hundred base pairs

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long, and at least the problem
becomes smaller.

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Maybe you could work it out.
Now, there were a couple of

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different ways of doing it.
One was by Wally Gilbert, who's up

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at Harvard who got half the Nobel
Prize for doing this.

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The other principle, the other one
that's proved to be most generally

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useful is Fred Sanger from England.
And he had Wally shared the Nobel

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Prize for discovering sequencing.
And the principal was disarmingly

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simple.  I think it's one of these
great ideas do you look back at

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afterward and think,
I could have thought of that.

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You guys already know everything
you need to invent how to sequence

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DNA.  I've told you all
the stuff already.

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But nobody's come down to tell me
that you've got it.

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And I didn't think of it.
So here is the principal.  What

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we've talked about,
if we take a DNA polymerase plus the

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four deoxynucleotide triphosphate's,
remember we talked about

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deoxyribonucleotide,
the adenosine triphosphate,

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and so on.  There's four different
ones.

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And we take a primer.
And there's a three prime hydroxyl

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right there.  And so this is the
other strand is going the opposite

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direction.  If we add that,
I think you all know what's going to

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happen.  We're going to get an
extension to the other end.

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And what happens every time we add a
nucleotide is that three prime

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hydroxyl attacks the phosphate of
the triphosphate.

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We lose two of the phosphates.
This is called pyrophosphate, and

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we've created a new five to three
prime linkage.

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That gives us a new three prime
hydroxyl, and we repeat the process,

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right?  That's what we talked about.
So, what would happen,

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let's spike in a little,
let me do it.  It's a little deoxy

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TTP.  So this is dideoxy.
But what would we mean by that?

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Well, if this,
remember where the deoxy came from?

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The ribose has at the two prime
position has a hydrogen instead of a

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hydroxyl, and at the three prime
position it has a hydroxyl.

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If we made a dideoxy, what we do is
we'd make that.  What could

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that nucleotide do?
Well, as long as the polymerase

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thought it was useful it would use
this end, it would have its

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triphosphate up here.
So, somebody else's three prime OH

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could come down and form a bond to
here and we'd lose this.

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So it could get incorporated.
That chain is finished.  It can't

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be elongated anymore.
So, let's think what would happen if

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we had, let me stretch this out a
little bit here,

00:15:09.100 --> 00:15:16.000
and let's imagine we had a few A's
in the sequence.

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So, we are just going to spike it a
bit.  So, most of the things will

00:15:22.100 --> 00:15:28.000
not see a dideoxy.
So, this primer will put,

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we'll try elongating this.
So when we get to this point,

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this point many of them will put it
an ordinary A,

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but a few will put it a dideoxy.
And those will finish.  At that

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point, they can't go any farther.
The rest of them keep going, [the

00:15:50.100 --> 00:15:55.000
various?] nucleotides.
When we get to the next A,

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most of them will put them a good T,
but the ones that put in a dideoxy

00:16:00.100 --> 00:16:06.000
will stop, and they will generate a
fragment that looks like that.

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You get the idea.
Out of this reaction,

00:16:11.100 --> 00:16:17.000
we are going to get a set of
fragments.  And each one terminates

00:16:17.100 --> 00:16:23.000
where there was an A up there.
Now, in this newer emulation of

00:16:23.100 --> 00:16:29.000
this thing, we have a T.
And the trick is to put a dye that

00:16:29.100 --> 00:16:35.000
you can attach to this nucleotide,
so it has a particular color.

00:16:35.100 --> 00:16:41.000
So, suppose we had something that
was yellow.  Then this particular

00:16:41.100 --> 00:16:47.000
set of fragments would be yellow.
And maybe you can begin to see what

00:16:47.100 --> 00:16:54.000
would happen now.
If we did the same game three more

00:16:54.100 --> 00:17:00.000
times, each time using a different
deoxy, next time maybe

00:17:00.100 --> 00:17:06.000
we'll use dideoxy A.
And we'll put a different colored

00:17:06.099 --> 00:17:11.000
dye on it.  Then every time,
in this case we come to a T in the

00:17:11.099 --> 00:17:16.000
template, it would stop,
and we'd get a little fragment

00:17:16.099 --> 00:17:21.000
that's stopped because it
incorporated a dideoxy A,

00:17:21.099 --> 00:17:26.000
and those would be, let's say,
green.  So, by the end of this,

00:17:26.099 --> 00:17:31.000
we would have all possible fragments
if we mixed them all together and

00:17:31.100 --> 00:17:37.000
the last nucleotide on each fragment
would say who it was by its color.

00:17:37.100 --> 00:17:40.000
So, if you were to,
then, take this whole mixture of DNA

00:17:40.100 --> 00:17:43.000
fragments and you run them down a
gel, in this case it's a difference

00:17:43.100 --> 00:17:46.000
of polyacrylamide gel because you
have smaller trying to get things to

00:17:46.100 --> 00:17:50.000
go by smaller fragments.
You could sort of see what would

00:17:50.100 --> 00:17:53.000
happen.  The big ones would be at
the top.  The small ones would be at

00:17:53.100 --> 00:17:56.000
the bottom.  And you'd see each band
would have a different color

00:17:56.100 --> 00:18:00.000
depending on the dideoxy that
terminated its chain.

00:18:00.100 --> 00:18:05.000
So, if you had a little scanner that
just goes along,

00:18:05.100 --> 00:18:10.000
it can read this,
and it will print out something.

00:18:10.100 --> 00:18:15.000
And these are always slightly
idealized.  This is a real one.

00:18:15.100 --> 00:18:20.000
But this is the sort of stuff you
get back.  If you send a piece of

00:18:20.100 --> 00:18:25.000
DNA over to a sequencing center,
they'd send this back as a file or

00:18:25.100 --> 00:18:30.000
something.  And you'd sit there.
And it's very good these days.

00:18:30.100 --> 00:18:34.000
The technology wasn't as good,
but they can almost always now get

00:18:34.100 --> 00:18:38.000
the sequence.  Occasionally,
you'll get something like a run of

00:18:38.100 --> 00:18:42.000
G's that gets a little hard,
but what they'll do is they'll sort

00:18:42.100 --> 00:18:46.000
of what they call sequencing [bow
strands?].  You can see this way,

00:18:46.100 --> 00:18:50.000
but really only looking at the
information of one strand.

00:18:50.100 --> 00:18:54.000
So, if we took the other strand.
So if we took the other strand, and

00:18:54.100 --> 00:18:58.000
we did the same thing,
but we should get the complementary

00:18:58.100 --> 00:19:02.000
piece of information.
So, what this DNA sequencing allows

00:19:02.100 --> 00:19:06.000
you to do, then,
is determine the exact sequence of

00:19:06.100 --> 00:19:10.000
nucleotides in some kind of piece.
And much of the art from the rest of

00:19:10.100 --> 00:19:13.000
it then comes,
how do you assemble all of those

00:19:13.100 --> 00:19:16.000
things together?
In the case of a bacteria or

00:19:16.100 --> 00:19:19.000
something, it wasn't so bad because
its DNA was small enough.

00:19:19.100 --> 00:19:23.000
You could cut it into a bunch of
sort of big fragments,

00:19:23.100 --> 00:19:26.000
and then take each one of those,
and then the sorting problem was

00:19:26.100 --> 00:19:29.000
relatively simple.
In the case of something like

00:19:29.100 --> 00:19:32.000
humans, it was really complicated
because there were

00:19:32.100 --> 00:19:35.000
so much more DNA.
And the other thing is higher

00:19:35.100 --> 00:19:39.000
organisms such as yourselves have a
lot of repeated DNA.

00:19:39.100 --> 00:19:42.000
It's just the same sequence,
and sometimes there's quite a bit of

00:19:42.100 --> 00:19:45.000
it, a bunch of repeats.
And so, if you see that at the end

00:19:45.100 --> 00:19:49.000
of your thing,
you don't really quite know where

00:19:49.100 --> 00:19:52.000
you are in the genome.
So a lot of other tricks had to be

00:19:52.100 --> 00:19:55.000
brought into play,
including knowledge of the human

00:19:55.100 --> 00:19:59.000
genetic map.  And so you could get
yourself anchored at various places

00:19:59.100 --> 00:20:02.000
because you do on this particular
piece of DNA, because it was

00:20:02.100 --> 00:20:06.000
associated with some gene,
had to be here on the chromosome.

00:20:06.100 --> 00:20:10.000
And therefore,
things at least decide beside it

00:20:10.100 --> 00:20:15.000
were there on the chromosome.
And there were a whole lot of

00:20:15.100 --> 00:20:20.000
tricks to putting it together.
But the very basic principle of how

00:20:20.100 --> 00:20:25.000
we sequence DNA has at its heart the
same process that I was talking to

00:20:25.100 --> 00:20:30.000
you about as when we were doing DNA
replication, except in this case

00:20:30.100 --> 00:20:35.000
it's just used in a very clever way.
And that was an amazing idea.

00:20:35.100 --> 00:20:39.000
It got a Nobel Prize,
and you've been sitting here for the

00:20:39.100 --> 00:20:43.000
last month with all the knowledge to
do it.  You keep emphasizing that

00:20:43.100 --> 00:20:47.000
you've got to have that three prime
hydroxyl.  But some of the great

00:20:47.100 --> 00:20:51.000
ideas often when you look back you
could see it was the hurdle was kind

00:20:51.100 --> 00:20:55.000
of small.  And they didn't even have
to do this with dyes at the

00:20:55.100 --> 00:20:59.000
beginning.  In fact,
that was a later innovation.

00:20:59.100 --> 00:21:03.000
The key thing was just the
dideoxies stopping in each place.

00:21:03.100 --> 00:21:11.000
I was lucky enough to live through
some of this, the development of

00:21:11.100 --> 00:21:20.000
this technology.
OK, so I've got one more really big

00:21:20.100 --> 00:21:28.000
thing to tell you,
which again was extraordinarily

00:21:28.100 --> 00:21:37.000
clever, but extraordinarily simple
once you heard about it.

00:21:37.100 --> 00:21:44.000
And it was one more technological
advance.  It wasn't a big insight

00:21:44.100 --> 00:21:51.000
into biology in and of itself,
but it was a technology that opened

00:21:51.100 --> 00:21:58.000
up just incredible experimental
possibilities.

00:21:58.100 --> 00:22:05.000
And it's something known as the
polymerase chain reaction.

00:22:05.100 --> 00:22:10.000
And this allows,
in principle, someone like me to go

00:22:10.100 --> 00:22:15.000
and to grab a single cell from you,
take it to DNA, and get a copy of

00:22:15.100 --> 00:22:20.000
any gene I want from your genome.
And I can look and see whether you

00:22:20.100 --> 00:22:26.000
have any mutations in that genome,
or whether there are different

00:22:26.100 --> 00:22:31.000
polymorphic alleles in the
population, in which one you've got

00:22:31.100 --> 00:22:37.000
from your mom,
or which one you got from your dad.

00:22:37.100 --> 00:22:41.000
So, you take from a single DNA
molecule, I can make as much as I

00:22:41.100 --> 00:22:45.000
want.  And this is just like DNA
sequencing.  You guys already know

00:22:45.100 --> 00:22:49.000
everything you need to know to
invent this technique as well.

00:22:49.100 --> 00:22:54.000
It has very much that same property.
It's another one of these very

00:22:54.100 --> 00:22:58.000
brilliant insights that you just had
to put things in the right place.

00:22:58.100 --> 00:23:03.000
So let me explain the principle.

00:23:03.100 --> 00:23:08.000
So, suppose that I would like to
know there's a gene that I know

00:23:08.100 --> 00:23:13.000
there's a family history of
something, and I would like to know,

00:23:13.100 --> 00:23:18.000
but I happen to get the allele that
carries that?  Or did I get the one

00:23:18.100 --> 00:23:23.000
that didn't?  So,
in principle what I would like to do

00:23:23.100 --> 00:23:28.000
is to get a hold of the piece of DNA
for that gene from my own cells.

00:23:28.100 --> 00:23:33.000
But all I've started with is my
entire DNA.

00:23:33.100 --> 00:23:36.000
Well, I could clone it.
I could make a recombinant library.

00:23:36.100 --> 00:23:40.000
I could do everything else.  But
there's this other simple way.

00:23:40.100 --> 00:23:44.000
And one way this involves, what it
involves taking,

00:23:44.100 --> 00:23:48.000
is since I know the sequence of the
genome now, I know that almost

00:23:48.100 --> 00:23:52.000
everything is going to the same.
There will be little differences

00:23:52.100 --> 00:23:56.000
between individuals.
I'll make a little primer that

00:23:56.100 --> 00:24:00.000
corresponds to the sequence that one
end of the gene,

00:24:00.100 --> 00:24:04.000
and another primer that corresponds
to the DNA at the other end of the

00:24:04.100 --> 00:24:08.000
gene, or whatever fragment
I want to use.

00:24:08.100 --> 00:24:12.000
And that's all I have to do in terms
of getting anything made.

00:24:12.100 --> 00:24:17.000
Now the rest, we are just going to
play games with DNA,

00:24:17.100 --> 00:24:22.000
with DNA polymerase, and nucleoside
triphosphates,

00:24:22.100 --> 00:24:27.000
just all the stuff I dragged you
through talking about DNA

00:24:27.100 --> 00:24:32.000
replication.  So here's the idea.
So here's my DNA, let's say, or

00:24:32.100 --> 00:24:37.000
part of it.
If I could actually see the sequence,

00:24:37.100 --> 00:24:41.000
I would know, let's say,
the gene I'm interested in is in

00:24:41.100 --> 00:24:45.000
here.  So, what I would do is make a
little primer.

00:24:45.100 --> 00:24:50.000
It just has to be enough to confer
specificity for something with

00:24:50.100 --> 00:24:54.000
humans.  If I make something
probably 30 nucleotides long,

00:24:54.100 --> 00:24:58.000
that's enough.  It'll only bind one
place in the DNA.

00:24:58.100 --> 00:25:03.000
And I make one, let's say,
for the opposite strand over here.

00:25:03.100 --> 00:25:15.000
So remember, this is five prime,
three prime, five prime to three

00:25:15.100 --> 00:25:27.000
prime.  So the principles will heat
to 95∞C, and will denature the DNA.

00:25:27.100 --> 00:25:39.000
And we'll add an excess of the two
primers.

00:25:39.100 --> 00:25:46.000
And let's say we'll cool to 55∞C,
or something.  And we'll cool it

00:25:46.100 --> 00:25:53.000
down enough so that we can get the
primers on.  But we are not going to

00:25:53.100 --> 00:26:00.000
go all the way and let all the
strands find their way back.

00:26:00.100 --> 00:26:08.000
And we'll add a DNA polymerase plus
four deoxy nucleoside

00:26:08.100 --> 00:26:15.000
triphosphates.
Well, what will happen?

00:26:15.100 --> 00:26:23.000
Well, here's one of the strands.
And we'll prime it here, let's say.

00:26:23.100 --> 00:26:31.000
So, it will copy down here and go
as far as it can go.

00:26:31.100 --> 00:26:38.000
And the other one starts here.
And it's going to go down all that

00:26:38.100 --> 00:26:44.000
way.  Let's just repeat the whole
process now, OK?

00:26:44.100 --> 00:26:50.000
What'll happen?  Now when we pull
them apart, we ought to have four

00:26:50.100 --> 00:26:56.000
strands.  We'll have the original
ones here, and when I repeat the

00:26:56.100 --> 00:27:02.000
process, the same thing's
going to happen again.

00:27:02.100 --> 00:27:09.000
This one will go here,
and it will copy out.  This one will

00:27:09.100 --> 00:27:17.000
go here.  It will copy out.
But what about this guy?  So,

00:27:17.100 --> 00:27:24.000
this one becomes this one here.
So the primer that it does will

00:27:24.100 --> 00:27:32.000
copy it, and it can't
go any further.

00:27:32.100 --> 00:27:37.000
I just generated a piece that's
exactly what I wanted.

00:27:37.100 --> 00:27:43.000
And the same deal here: as long as
I don't get lost,

00:27:43.100 --> 00:27:48.000
which what did I do?
So, we've got this guy here.

00:27:48.100 --> 00:27:54.000
So, it starts there.  So this one
becomes this one,

00:27:54.100 --> 00:27:59.000
and we'll prime it here.
It'll go along and it will stop.

00:27:59.100 --> 00:28:05.000
So there's the complementary strand
to the one here.

00:28:05.100 --> 00:28:09.000
And I think this is sort of like
doing a math problem.

00:28:09.100 --> 00:28:14.000
You can't just look at it and say,
we'll maybe you will get it.  But

00:28:14.100 --> 00:28:19.000
there's nothing like sitting down
with a pencil and paper,

00:28:19.100 --> 00:28:24.000
and take yourself through several
cycles.  What you will believe is

00:28:24.100 --> 00:28:29.000
how quickly you get to get being
nothing but, almost nothing,

00:28:29.100 --> 00:28:34.000
but the sequence that you are trying
to amplify.

00:28:34.100 --> 00:28:38.000
And so, this again has an
astonishing effect.

00:28:38.100 --> 00:28:42.000
This is why you hear about DNA
testing all the time in forensics,

00:28:42.100 --> 00:28:46.000
because you can take a tiny bit of
DNA from saliva,

00:28:46.100 --> 00:28:50.000
or semen, or blood,
or whatever they might find on a

00:28:50.100 --> 00:28:54.000
crime scene, and then they can
amplify little pieces

00:28:54.100 --> 00:28:58.000
and they compare.
And there's a trick they use in

00:28:58.100 --> 00:29:03.000
forensics, and that is that there
are sequences within the human

00:29:03.100 --> 00:29:08.000
genome where the little variable
repeats like GT,

00:29:08.100 --> 00:29:13.000
GT, GT, GT, GT, GT,
and I might have 14 of them in one

00:29:13.100 --> 00:29:18.000
of my chromosomes.
The one I got from my mom might

00:29:18.100 --> 00:29:23.000
have 40.  You might have 24 and
something else,

00:29:23.100 --> 00:29:28.000
and so on.  If you were to do PCR
around a little region that was

00:29:28.100 --> 00:29:33.000
known to be variable,
if you had 14 repeats you'd get a

00:29:33.100 --> 00:29:38.000
shorter fragment.
And if you had 40 repeats,

00:29:38.100 --> 00:29:42.000
you get a longer fragment.  So,
I'll come back to that in a sec.

00:29:42.100 --> 00:29:47.000
So, if you were to, for example,
take something with a long [repeat

00:29:47.100 --> 00:29:52.000
and a?] short peak into this kind of
thing.  We get two fragments,

00:29:52.100 --> 00:29:56.000
say, one from the paternal.  And if
you do this with several such sites

00:29:56.100 --> 00:30:01.000
around the genome,
pretty soon you run into situations

00:30:01.100 --> 00:30:05.000
where the odds of a particular
combination of a long one at the

00:30:05.100 --> 00:30:10.000
site, a short one at the site,
and so on, becomes statistically

00:30:10.100 --> 00:30:15.000
improbable that it's anyone
other than yourself.

00:30:15.100 --> 00:30:18.000
So on a crime scene,
if they did this, they,

00:30:18.100 --> 00:30:21.000
for example, might have three
individuals that they were thinking

00:30:21.100 --> 00:30:24.000
was possible.  And they'd generate
patterns like this,

00:30:24.100 --> 00:30:28.000
say, using three different loci like
this, and then have the

00:30:28.100 --> 00:30:31.000
forensic sample.
And it was pretty evident who didn't

00:30:31.100 --> 00:30:35.000
do it, and who at least remains a
suspect.  This probably would

00:30:35.100 --> 00:30:39.000
improve it.  The very last thing,
just to close us off, is when people

00:30:39.100 --> 00:30:42.000
develop this PCR technique,
you had to sit there with your

00:30:42.100 --> 00:30:46.000
pipette because every time you
raised it to 90∞ to denature the DNA

00:30:46.100 --> 00:30:50.000
you killed your enzyme.
So, you cool it down to 55,

00:30:50.100 --> 00:30:53.000
escort it in a new DNA polymerase.
And then someone finally said,

00:30:53.100 --> 00:30:57.000
another brilliant idea,
what if I had a thermoresistant

00:30:57.100 --> 00:31:01.000
polymerase?  Where would
I find those?

00:31:01.100 --> 00:31:04.000
Well, Penny was talking to you about
those events where it's really,

00:31:04.100 --> 00:31:08.000
really hot, and those black smokers
and everything,

00:31:08.100 --> 00:31:11.000
so maybe you got a bacterium from
their.  It would have a temperature

00:31:11.100 --> 00:31:15.000
resistant polymerase.
So here you are from the New

00:31:15.100 --> 00:31:19.000
England [Biocatalog?
, [vent exominus?] DNA polymerase,

00:31:19.100 --> 00:31:22.000
deep vent DNA polymerase.  People
went to grab those bacteria from

00:31:22.100 --> 00:31:26.000
there, grabbed the DNA polymerase
gene.  And now,

00:31:26.100 --> 00:31:30.000
the DNA polymerase just sits there.
It just laughs and you bring it up

00:31:30.100 --> 00:31:34.000
to 90∞.
And when you cool it back down and

00:31:34.100 --> 00:31:38.000
give it a substrate again,
it will do its thing.  And so,

00:31:38.100 --> 00:31:43.000
this whole thing can be done
automatic and you don't have to sit

00:31:43.100 --> 00:31:47.000
there and pipette something in at
the end of every run,

00:31:47.100 --> 00:31:52.000
another little cute sort of
engineering trick that combined

00:31:52.100 --> 00:31:55.000
ecology together with biology.
OK, see you on Friday.