The following content is
provided under a Creative
Commons license.
Your support will help MIT
OpenCourseWare continue to
offer high quality educational
resources for free.
To make a donation or view
additional materials from
hundreds of MIT courses, visit
MIT OpenCourseWare at
ocw.mit.edu.
PROFESSOR: Good morning.
AUDIENCE: Good morning.
PROFESSOR: All right.
So today--
I haven't seen you in a while.
Anyway, today, we're going to
turn back to our picture,
function gene protein.
We filled in genetics.
We filled in biochemistry.
We've now got the connection
between gene and protein
through molecular biology.
We know gene encodes protein.
We know central dogma,
DNA goes to the
RNA goes to the protein.
We know all that in theory.
In fact, people knew this by
the middle of the 1960s.
People were so excited that they
understood the idea of
how genes give rise to proteins
through transcription
and translation, they read the
genetic code, that they
declared victory.
Some of them said, done with
the secret of life.
Let's go on and do the brain.
That was actually the thinking
of a lot of the great
molecular biologists
in the late 1960s.
Let's go do the brain.
Why did they say such a thing?
Well, because they
thought they were
done with the problem.
They thought that once you knew
in principle how a gene
gave rise to a protein,
you could do it.
But in practice, nobody could
read a single gene.
Nobody could even identify
a single gene.
Maybe that's why they went on
to say, let's go study the
brain, because they actually
weren't sure what they could
do past that point.
All right.
So wait a second,
wait a second.
I said nobody could even
purify a single gene.
Didn't we talk about purifying
the genetic material?
You're supposed to say
yes at that point.
Yes, right?
We talked about that.
Avery, McCarty, MacLeod-- we
purified the genetic material.
We did it by using this
assay of transforming.
So what do I mean by we can't
purify a single gene?
What I mean is that we can
purify the hereditary material
away from everything else, but
we get all of it together.
We don't get individual genes
separated from each other.
We get the whole mixture
of all the genes,
all the genetic material.
As a biochemist, how are we
going to ever separate the
gene encoding--
oh I don't know, ARG1, our
favorite gene for arginine
biosynthesis--
from the gene encoding
ARG2, for example?
What kind of biochemistry
can we do to
separate these two genes?
Do they have different
biochemical properties?
What's so different about
ARG1 and ARG2?
What's their different
biochemical properties?
They're both DNA.
They have exactly the same
building blocks, slightly
different order.
You think you have a
purification procedure, I'm
going to run it over some column
and separate it by
something that's going to
separate ARG1 from ARG2?
No.
From the point of view of a
pure biochemist, they look
exactly the same.
All the different genes have
the same biochemical
properties.
How in the world would we ever
purify ARG1 from ARG2, or in
the human, the gene encoding
hemoglobin from the gene
encoding collagen from the gene
encoding keratin from the
gene encoding anything else?
Think about it.
That's a tough problem.
There is a brilliant solution
that arose in the 1970s to how
we could purify the individual
genes away from each other.
But it's like no other piece
of biochemistry anybody had
ever seen before.
It has a totally different
principle behind it.
Because it isn't just
fractionating things according
to their biochemical
properties.
It involves something else.
And it's called cloning.
It is called cloning.
Molecular cloning.
You see, the problem is this.
The human genome--
how big is the human genome?
How many bases?
Three billion bases, three
times 10 to the
ninth bases, right?
How big is a typical
human gene?
A typical human gene might
be 30,000 bases.
How big is a typical mutation
that we might want to find in
a typical gene, like causing
sickle cell anemia?
One base.
We've got to purify out genes
that are one part in 10 to the
fifth and mutations that
are one part in 10 to
the ninth or so.
And how are we going
to do that?
Well, the trick is this.
I'll give you the quick
overview, and then we'll spend
today looking at it.
Step one is we cut up our DNA.
We cut DNA at defined sites, and
we then paste the DNA to
distinct molecules
called vectors.
These vectors have a cool
property, that when you take a
vector and you insert in it a
piece of DNA, that vector is
able to grow in another
organism.
You then transform the DNA--
that's transfer, we use the
word transform the DNA--
into something like E. coli,
where you get your little
vector in there.
It grows within E. coli, and as
E. coli divides, it makes
copies of itself.
And then you select those
bacteria that have received,
that have been transformed, grow
them up on a petri plate
so that you have little
colonies.
And then you screen
the colonies.
Now, what do I mean?
We cut the DNA.
We paste the DNA.
We transform the DNA.
We select the bacteria that
have been successfully
transformed.
And we screen the resulting
colonies to find what we're
looking for.
Now, notice--
that amazing trick here is when
we cut up the DNA into
single molecules, lots and lots
of single molecules, and
we paste them into vectors,
and we transform them into
bacteria, each one of those
bacteria gets exactly one
molecule, give or take.
It gets one piece
of human DNA.
We then spread them out on a
plate and they grow up, and
each one grows up
copies for us of
individual pieces of DNA.
That is so cool.
Because we've just accomplished
biochemical
purification.
It's not based on any different
properties of the
individual molecules.
It's based on the fact that
we dilute them, in effect.
They're diluted, and
one molecule
ends up in each bacteria.
So they're purified
in that sense.
And then when that bacteria
grows up, everything it grows
up is a pure copy, a copy of
that single piece of DNA that
went into it.
That's a different kind
of purification.
When I'm done-- and we'll go
through this whole process.
That's the point of today's
lecture, is to go through the
whole process.
When I'm done, I have
bacteria spread out.
And right over there, one of
these guys has ARG1, and one
of these guys has ARG2, and one
of these guys has ARG10.
Now, admittedly, I don't know
which one has which, but I've
accomplished the purification.
I'll then have to figure out
how to screen and find out
which one is which, but I have
separated the molecules away
from each other by this process
of cloning, diluting
them in a way--
one molecule per bacteria--
and growing them back up.
I could do this for anything.
I could dilute proteins down
into test tubes that had one
protein molecule per test tube,
and I could say I've
accomplished purification.
The problem with it is I have
no way to replicate those
proteins to get a meaningful
amount of it.
But when it's DNA, and I've put
it back into a bacteria, I
have a way to grow it back up.
And that's why this
trick works--
is because DNA is a molecule
that can replicate.
No other molecule has
that cool property.
And so you can pull
this off for DNA.
All right.
Now we have to dive in to
understand how this could
possibly work.
