PROFESSOR: So, first step,
we need to cut our DNA.
Step one, cut, which is going to
be DNA restriction enzymes.
It turns out, quite remarkably,
that if I have a
sequence of DNA, five prime A G
C T A G A A T T C T T A C C
three prime, and we'll
come backwards
filling in the sequence.
It turns out that molecular
biologists are so cool that
they invented a protein that is
able to recognize that six
letter sequence, G-A-A-T-T-C.
And it is able-- actually it's
G-A-A-T-T-C on this strand,
but what is it coming back?
It's G-A-A-T-T-C. It's
the same thing.
So actually this is
a palindrome.
That's kind of nice.
It's a palindrome--
it's a reverse palindrome.
It's the same word spelled
backwards on the other strand.
So what it does is it
cuts it like that.
And what it produces is a DNA
molecule like this and a DNA
molecule like that, that's
mostly double stranded, but
has a four base pair overhang.
The overhang reads
T-T-A-A here.
It reads A-A-T-T there.
And remember this is five prime
to three prime, five
prime to three prime.
And there you go.
This guy has its little
phosphate at the end there.
This guy has his little
hydroxyl over there.
And it cuts it.
Now that is an incredible
piece of engineering.
To come up with a protein, to
devise a protein, that is able
to recognize those six bases
and cut at those six bases.
And cut in just this
way making a really
clean overhang here.
It's this cool five
prime overhang.
Who do you think invented
this cool protein?
What engineer came up with
this cool protein?
AUDIENCE: MIT engineers.
PROFESSOR: MIT engineers,
yeah.
Not a chance.
Not a chance.
This is a really tough feat.
This is something that can only
be done by the smartest
engineers on the planet.
And MIT engineers are
unfortunately only the
smartest human engineers
on the planet.
Who came up with this
is E. coli.
AUDIENCE: So you found it
somewhere in nature?
PROFESSOR: Sorry?
AUDIENCE: You found it
somewhere in nature?
PROFESSOR: Of course you find
it somewhere in nature.
Almost everything important
that we say molecular
biologists have come up with, it
means molecular sat at the
feet of the true masters,
bacteria, and learned from the
true masters.
This protein is found
in nature.
And it's found in E. coli.
In fact, it's found in E. coli
strain R. And it was the first
such protein found in E.
coli strain R, so it
gets the name EcoR1.
And it cuts the DNA like this.
Pretty cool.
Pretty cool.
Now, it turns out that E.
coli has this EcoR1.
How often does E. coli--
so whenever I take EcoR1, this
protein, purified from E.
coli, and I add it to DNA it
always cuts at this site,
which we call an EcoR1 site.
How frequently do we
expect, what's the
frequency of EcoR1 sites?
G-A-A-T-T-C, how often will
that occur at random?
One in--
AUDIENCE: Two to the sixth?
PROFESSOR: One in two
to the sixth?
How many letters do I have?
AUDIENCE: Four, oh,
four to the sixth.
PROFESSOR: One in four
to the sixth.
My frequency should be about
one in four to the sixth,
which is about what?
What's four to the sixth?
It's two to the 12th.
It's about 4,000.
It's about one in
4,000 letters.
One in 4,000 bases.
So it's very convenient.
One in every 4,000 bases
it'll roughly cut.
It'll cut at roughly
one 4,000 bases.
Why doesn't E. coli
cut its own DNA?
If it's got this protein
floating around in its cell,
why isn't it chopping
up its own DNA?
Doesn't have G-A-A-T-T-C?
Yeah, the problem is
it's so frequent.
That'd be really hard
to make sure--
I mean, E. coli has 4 million
letters in its genome.
This should cut every
4,000 bases.
You expect about 1,000
such sequences.
It might be hard to arrange
not to cut--
not to have any such
sequences.
It's a good idea, is not to have
any, but an alternative--
AUDIENCE: [INAUDIBLE].
PROFESSOR: It protects them.
It turns out E. coli, instead
of avoiding the sequence
altogether, has another
trick up its sleeve.
E. coli protects this sequence
whenever it occurs.
So it turns out that whenever
you have a stretch of the E.
coli genome that has this
G-A-A-T-T-C in it, what E.
coli does is it puts--
I'm just writing M-E here
for a methyl group.
Right, C-H three up there.
It puts a methyl group--
I'll write C-H three.
There we go.
It puts a methyl group on
the A, that middle A.
Well, that is a cute trick that
E. coli uses, putting a
methyl group there.
Because what happens is, when
there's a methyl group, right
at that position, the enzyme
no longer recognizes and no
longer cuts there.
So that's kind of clever.
E. coli makes this protein
that can recognize
G-A-A-T-T-C, but it has a
second protein that puts
methyl groups there.
And this protein happens
by accident
to be called a methylase.
It has a methylase.
And the methylase protects
that sequence.
So now, this is really cool
engineering, but kind of dumb.
What's it doing there?
It has something that cuts the
sequence and it protects the
sequence, why bother
having this?
Yeah?
AUDIENCE: You can use it to cut
it at places to unwrap the
true strands.
PROFESSOR: That's an
interesting idea.
We could use it to cut our DNA
and open it up to unravel our
true strands.
It's a thought.
Yes?
AUDIENCE: To protect the
bacteria from viruses?
PROFESSOR: Protect the bacteria
from viruses.
How do you protect yourself
from viruses?
Well, you have an immune system
with immune cells and
antibodies and all that.
Does E. coli have an
immune system?
Why doesn't it have
immune cells?
Because it's like one cell.
How's it going to have an
immune system, right?
So suppose E. coli
gets a cold.
Suppose it gets infected
by a virus.
How's it going to
protect itself?
Cut at a frequently occurring
DNA sequence.
Now the virus, of course, isn't
methylated there, bingo.
That's how it tells its own--
you can tell cell
from an invader.
E. coli can tell cell from
an invader because it's
methylated its own G-A-A-T-T-C
sites, but the virus isn't
methylated there.
Way cool.
This is an immune system
for E. coli.
Now, it turns out-- so
this is protection.
These restriction enzymes
protect E. coli from viruses.
It turns out that E.
coli is not alone
in this clever trick.
It turns out that other bacteria
have also thought of
this trick.
So it turns out that there is
another restriction enzyme
that cuts at G-G-A-T-C-C. And
on the other strand it goes
G-G-A-T-C-C. It, again, cuts in
that distinctive pattern.
And it's called BamH1.
And there's another guy.
And he cuts at A-A-G-C-T-T,
A-A-G-C-T-T. And it
also cuts like that.
And it's called HindIII.
And there's some that
cut at G-A-T-C, just
the four letter word.
And they cut like that.
And there's some that cut at
C-A-G-C-T-G, C-A-G-C-T-G, and
this, cuts smack
in the middle.
In other words, there's a wide
number of different tricks.
Some cut at six bases.
Some cut at four bases.
Some cut at eight bases.
Some cut leaving an overhang.
Some cut smack in the middle.
Some cut leaving the overhang
in the other direction.
Some allow a degenerate
base in the middle.
It doesn't care which base
is in the middle.
There's a zillion different
solutions that bacteria have
come up with for their
immune system.
And so, if I want to cut up some
human DNA all I need is
say, this protein EcoR1 or BamH1
or HindIII or MVL 1 or
PVU 2 or et cetera.
And I can do that by growing
up E. coli and
purifying the protein.
And if I wanted HindIII, I
would grow up haemophilus
influenza and purify
the protein.
So in a molecular biology lab,
today, if you want to cut up
human DNA, you could grow up
some E. coli and purify EcoR1
or haemophilus influenza.
And that is indeed what ancient
molecular biologists
did in prehistoric days in
the 1970s and 1980s.
They would purify their own
restriction enzymes.
They're still alive today.
You can talk to them.
There are many of them
on the faculty.
And they'll tell you how it put
hair on their chest to be
able to purify their own
restriction enzymes.
What do you do today?
Order it online from
the catalog, right?
You know, there's the
catalog, the New
England Bio Labs catalog.
Let's see what we got here.
Restriction enzymes, modifying
enzymes, polymerases, all
right, EcoR1, sale on
EcoR1 right now.
$100 buys you 10,000
units of EcoR1.
It's in the catalog.
You can go online.
You can order it.
You can have it tomorrow
by FedEx.
So, but that's how it works.
It's in the catalog.
So you can get any restriction
enzyme you want to cut DNA
anywhere you want to.
