Overview of Recombinant DNA, excerpt 1 | MIT 7.01SC Fundamentals of Biology


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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.

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