HAZEL SIVE: Welcome back to
Getting Up to Speed in Biology.
Today is our last discussion.
And the topic today
is building with DNA.
We're going to cover
four topics today.
And I think you will find them
both interesting and useful.
The first is
genetic engineering.
I'll talk about what that is.
We'll then talk about
something called
restriction endonucleases.
We'll talk about
vectors and ligation.
And then we'll talk
about something
that has the abbreviation PCR.
Let's start with the topic
of genetic engineering.
I bet you've heard of this.
Another word for
genetic engineering
is recombinant DNA technology,
recombinant DNA technology.
And what it really means
is making DNA constructions
in the lab.
Building with DNA, the title
of our discussion today,
DNA construction in the lab--
something that people do
to build DNA molecules.
And one of the aspects
of genetic engineering,
the subtopic that
is really crucial,
is called molecular cloning.
And that refers to making
lots of the same DNA,
making lots of the same DNA.
And lots of the same
DNA or molecules of DNA
that are all very similar
to one another or identical
are called clones.
Now, why would you
want to do that?
Let's take a moment
to look at some
of the power of
genetic engineering
and give you a sense of why we
are even having this discussion
today and why this is
part of your getting up
to speed in biology.
Genetic engineering
is everywhere.
There are
genetically-modified animals.
Some of them are kind
of fun, like fish
that glow in the dark
different colors.
Some of them are for
research like mice
that have been genetically
engineered to glow green
or to glow green in
different tissues.
Some of them are
for food, cattle
that have been engineered
so that they produce
much leaner meat or chickens
that have been engineered
so they don't have any feathers
and are easier to process
after they have been killed.
Plants are genetically
engineered,
again, some of them for
food, for example rice, which
is one of the staple
crops of our planet,
lacks a specific substance
called beta carotene.
And beta carotene is required
for vitamin A production, which
is essential for life.
You can genetically
engineer rice
so that it produces
beta carotene.
And this is not scary.
It's just useful.
And then finally, there
is a lot of health use
of genetic engineering.
One can manufacture
particular human proteins
by genetic engineering,
for example, insulin.
One can think about
going and curing
human diseases that are
caused by variant genes that
have bad effects,
deleterious effects,
maybe even by replacing
the faulty gene
with the normal
gene, although that
is right out there
at the limits of what
is ethically permissible.
And it's presently not something
that we're doing in humans.
But genetic engineering
is everywhere.
It is really interesting.
It is really cool.
And it is really fundamental
to biology today.
Let us think therefore how
you do genetic engineering.
That's what we're going
to talk about today.
How do you build with DNA?
And let's consider the kind
of global process of building
a new DNA molecule with the
use of genetic engineering
techniques, and then we'll
go through step by step
how you do each of
the steps involved.
Here then are some basic steps
of building a recombinant DNA
molecule where the
term recombinant means
that the DNA comes from
two or more sources.
The first thing
that you need to do
is to think about what you're
going to engineer, obviously.
And you'll usually have
some gene of interest,
which we'll abbreviate GOI.
You're going to
isolate the DNA that
encodes the gene of
interest or part of it.
And to do that, you usually
cut out the gene of interest
from some larger piece of DNA.
Remember, we talked
about chromosomes
being long strings of genes and
other DNA all joined together.
If you're going to get your
gene of interest or part of it,
you have to be able to isolate
it from the chromosome.
So that is one of the
first steps, this cutting
out of the gene of interest.
And then you want to take
your gene of interest
and put it in some
environment so
that you can grow lots of it.
And the way you do
that is to paste it--
paste your gene of interest--
into a vector, where
a vector is a carrier.
A vector is a carrier
of DNA, of extra DNA.
It itself is DNA.
And it has the property that
it replicates very avidly.
And so you can grow
lots of your clone.
So a vector is a carrier of
extra DNA that replicates.
And because it
replicates, you can
get lots of your clone
DNA, lots of clone DNA.
That's the basic process.
And what we're going
to talk about today
are two parts of this, really.
We're going to talk
about cutting out
your gene of interest.
And we're going to talk about
pasting your gene of interest
into a vector.
If you look at the schematic
that I drew for you,
the different steps are shown
here starting off with cells.
We open up the cells.
We break them open.
We extract the DNA.
We cut and isolate
the gene of interest,
and then we insert or paste the
gene of interest into a vector.
That gives us a
recombinant clone.
Because the vector
can replicate,
we can grow a lot of
gene of interest DNA,
and then we can do
things with that DNA.
It can be used to make
RNA, which then can then
be translated into a
protein of interest.
And that protein of
interest, maybe insulin,
can then be purified in the
lab and modified if necessary
and used as a human therapeutic.
We're going to start by
talking about cutting the DNA.
And that is done
with reagents called
restriction endonucleases,
restriction endonucleases.
These are enzymes.
They're proteins.
And they're enzymes,
biological catalysts,
that precisely cut DNA.
Sometimes, they're called
molecular scissors.
And they do so because
the proteins that
are the restriction
endonucleases
recognize particular
DNA sequences, sequences
of bases in the DNA.
They bind to those sequences,
and then they have activities
so they can break the
double-stranded DNA
in very precise ways.
Let's write that down.
These are enzymes that
precisely cut DNA.
They recognize.
They bind, and they cut
specific DNA sequences.
And once they've
done their cutting,
there are two types of
ends of the cut DNA.
These ends are
either called blunt
or they are called sticky.
And you're going to need
to know the difference.
And we'll follow through this
on the next couple of boards.
Let's start off by talking about
blunt restriction endonucleases
and blunt ends after restriction
enzyme or endonuclease cutting.
And we'll use, for example,
an enzyme that is called SmaI.
So, blunt-ended
restriction endonucleases,
I'm going to abbreviate
restriction endonuclease RE.
And the example I'll use
is S, big S, small maI.
Sma1 refers to a
particular bacterium
from which these
restriction endonucleases--
this restriction
endonuclease-- was isolated.
Restriction enzymes are part
of the bacterial defense system
so that if the
bacterium is invaded
by a virus that is a
DNA virus, these enzymes
will cut up the viral DNA
and render it nonpathogenic.
And now we use these enzymes
for genetic engineering.
The SmaI recognition
site looks like this--
CCC GGG 5 prime to 3
prime on the top strand.
And you know now what
the complementary strand
looks like.
It is 3 prime GGG CCC 5 prime.
When SmaI recognizes this
site, it makes two cuts.
It cuts one strand here
and one strand opposite.
We can also indicate these by
arrows and a vertical line.
There are a number of
different notations
you can use to indicate where
the restriction enzyme cuts.
And out of this come
two DNA fragments.
One of them looks
like 5 prime CCC 3
prime, and on the other
strand, of course, GGG 3 prime
to 5 prime antiparallel.
And the other fragment
looks like 5 prime GGG 3
prime and 3 prime CCC 5 prime.
You can see that the ends
of the two pieces are flush.
There's no nucleotide
sticking out,
no base sticking
out on either end.
That's why this is
called a blunt cutter,
a blunt-ended restriction
enzyme cutter.
So both of these are made.
You can also see that the
SmaI site is a palindrome.
This is the recognition
site to SmaI.
It is a palindrome.
And many restriction
recognition sites are.
Good.
Let's look at a
sticky-ended site now.
So a sticky end
restriction enzyme.
And the example we'll
use here is called EcoRI.
And it has a
recognition site that
goes 5 prime GAATC 3 prime
and on its other strand CTTAAG
5 prime.
You can see now one
of the many reasons
you need to know about
complementarity is
so you can easily fill
in the other strand.
EcoRI cuts in a
different way than SmaI.
It cuts off this
G on that strand
and the matching G
on the other strand.
And we can put an
arrow here if we want.
And I sometimes put a
line right across where
the cut is going to be made.
And now we have to figure
out after the cut what
are the two pieces of DNA
that are made look like.
And the easiest way to do
this is start at the cut
either at the arrow or your
slash line and kind of peel
away the DNA until
you get to the place
where the other strand is cut.
So let's draw this now.
We'll have 5 prime
G on the one strand.
And on the other strand,
we'll have CTTAA.
5 prime, 3 prime--
never forget to put in those
5 primes and those 3 primes.
That's what the piece
from the top looks like.
How about the piece
from the bottom?
There, we have 5
prime AATTC 3 prime.
And we have on the other
strand a G. That's what we get.
These ends are sticky because
there is single-stranded DNA
that is left after the cut.
And the single-stranded
DNA is able to base pair.
And it is therefore sticky
with regard to its ability
to base pair.
That's where the term
"sticky end" comes from.
So here again is the
recognition site for EcoRI.
And again it's a palindrome.
EcoRI leaves a 5 prime overhang.
That means that there is more
DNA towards the 5 prime end
than the 3 prime end
in the sticky cut.
There are also enzymes--
and I'll show you on
a slide in a moment--
that leave a 3 prime overhang.
So this region here
is a 5 prime overhang.
And there are-- I'll make the
note-- there are both 5 prime
and 3 prime overhang
sticky-ended restriction
endonucleases, REs.
Let's take a look at a
couple of slides here.
There are thousands of
restriction endonucleases.
As I mentioned,
they were all named
after the bacterium
from which they
were isolated, so EcoRI from
E. coli, PstI from Providencia,
and so on.
You can see the double-stranded
recognition site
in the DNA and, on this
diagram, the structure
of the cleaved products.
You can look through books
of restriction endonucleases.
And you can find something
that cuts exactly where you
want it to in the DNA.
And you can get therefore a very
precise cut in a piece of DNA
when you want to
do a cut and paste
to make a recombinant clone.
I've redrawn for you the
SmaI, SmaI or EcoRI sites.
And then on the
bottom of the slide,
I've also put in another
site which leaves
sticky 3 prime overhang ends.
And this is PstI.
And you can see
here that there is
extra unpaired single-stranded
DNA sticking out
towards the 3 prime end of each
of the ends of the cut DNA.
One thing I want to emphasize
is that these restriction
endonuclease sites are in
a whole big piece of DNA.
We draw them just as the six
base pair or the four base
pair or the eight
base pair cut site.
But of course, they are
joined to long pieces of DNA
on either side.
Good.
Now you've had some
information about restriction
endonucleases, I want you to
go to the class assignment
and practice doing some
restriction enzyme work.
