HAZEL SIVE: From your class
exercise about restriction
endonucleases, you should
now be able to manipulate
a piece of DNA to reveal
blunt ends or sticky ends.
You should know whether or
not it's a 5-prime overhang
or a 3-prime overhang.
And this will set us up for the
next topic I want to discuss,
which is the question of
the vector and ligation.
Let's set the stage
for this topic
by looking back at
the overall view
that I gave you on cloning
and using a gene of interest.
We've talked about
cutting the DNA now.
And we haven't talked
about how you exactly
isolate your gene of interest.
But we'll not worry about
that for the moment.
What we need to talk about
now is the next step,
how you're going to put
your gene of interest
into some kind of
carrier DNA molecule that
will allow it to replicate
to high copy number.
And the reason
that this can occur
is because of these
things called vectors.
A vector is really a virus
that grows in bacteria.
It may be pathogenic, or it may
be harmless to the bacterium.
The vector is made of DNA.
And it is often
circular, in which case
it has got a special
name, which is a plasmid.
The trick about
plasmids, and any vector,
is that they have
something called
an origin of replication.
This is abbreviated ORI.
And this origin in the vector--
I'll write it out--
is an origin of replication.
And this refers to
DNA replication.
The origin is a
particular sequence
in the plasmid that tells
DNA replication to begin.
It is a start site
for DNA synthesis.
In fact, it's not just
plasmids and vectors
that have got origins
of replication.
Our own chromosomes do, too.
I just didn't tell
you about them
when we were talking
about DNA synthesis.
But now you've
heard of them, you
can add that to your
compendium of knowledge
about DNA synthesis in general.
This origin of
replication allows
the plasmid to replicate
within the bacterial host
cell to very high copy
number, 10,000 copies or more.
And because you could
grow billions and billions
of bacteria without
too much difficulty,
you can land up with very
large amounts of DNA--
grams, even kilograms of
DNA from the right amount
of bacteria that are
growing the viral vector.
OK.
So the origin of replication
allows the vector
to replicate to something
like 10,000 copies per cell.
And that will give
you lots of DNA.
There's one other property
that vectors usually have.
And that is called
a selectable marker,
or two selectable markers.
And I'll tell you what
they are in a moment.
But let's just put
down that vectors also
have a selectable marker.
There's our vector.
What we have to do now is to
insert the gene of interest
into the vector.
We have to do a pasting.
And we paste the
gene of interest
into the vector using a
particular enzyme called
DNA ligase.
Our gene of interest is
pasted, or covalently bonded,
through phosphodiester
bonds that
join up the nucleic acid polymer
is pasted or covalently bonded
into the vector using an
enzyme called DNA ligase.
And just a little note--
you heard about restriction
endonuclease and DNA ligase.
If you hear about a molecule
that's got the "-ase"
at the end, it's
usually an enzyme.
What is this DNA ligase?
It will join together any
two compatible DNA ends, OK?
What does that mean?
So ligase joins two
compatible DNA ends.
And now, you have to think
back to the restriction
endonuclease ends.
Any two blunt ends
can be joined together
because there's bluntness there.
There's no kind of nucleic
acid hybridization involved.
Any two blunt ends, restriction
enzyme endonuclease ends
can ligate.
But the same thing is
not true of sticky ends.
There, you've got the single
stranded bits sticking out.
That's why they're sticky.
And in order to get
sticky ends to ligate,
those sticky single
stranded bits
have to base pair
with one another.
If they can base pair
exactly, then ligase
will join them together and make
a covalently closed molecule.
So two complementary, or
ends that can base pair,
those complementary
ends can ligate.
After ligation of restriction
endonuclease cut DNA,
you may or may not regenerate a
restriction endonuclease site,
OK?
That's not part of the deal.
So you may or may not regenerate
a restriction endonuclease
site.
Let's look at a
couple of slides,
so that you can see what I mean.
Here, firstly, is your
basic plasmid vector.
This one, called pBR322,
was one of the very first
that was used in
genetic engineering.
You can see a region called ORI.
That is the origin
of replication.
And it's where DNA
replication starts.
Both the vector and the gene of
interest that you insert in it
will be replicated, starting
at this origin of replication.
And then there are
these selectable markers
that we mentioned, that are
here listed amp and tet.
Now, let's look at
some compatible ends.
Any blunt ends can ligate.
I just made up two in
the top of the slide.
One of them is half
of an SmaI site,
but the other one
is a different site.
But they can ligate
just fine because all
you need are 3-prime and
5-prime ends to join together.
You need the 3-prime hydroxl
and the 5-prime phosphate.
And then ligase can come along.
And it can make a phosphodiester
bond between those two
molecules and join them
back together again.
Any complementary
sticky ends can ligate.
So complementary means
that they can base pair.
So for example, in the
example of the EcoRI site,
you can see that if you
take two 1/2 EcoRI sites
and put them back
together again,
that they will be able
to ligate and regenerate
a covalently closed DNA
molecule and regenerate an EcoRI
site, OK?
The base pairing is exact there.
The AATT on this
molecule will base pair
with the TTAA on that molecule.
And you will get out the
perfect covalently closed DNA.
On the bottom of
the slide, you'll
see that there are two
compatible ends there.
They have, again, got
the AATT TTAA base
pairing that can make a closed
DNA molecule that ligase
can work with.
But in fact, the outcome
is not a restriction site.
It's not an EcoRI site.
There is an AT on the 5-prime
end of the top strand,
and then the 3-prime end
of the bottom strand.
And that doesn't give
you an EcoRI site, OK?
So that's some detail
about compatible ends.
And you will need to be
able to work with those
in order to figure out whether
or not your gene of interest
can ligate into your vector.
Let's complete the
process of thinking
about now how to get the gene
of interest into the vector, OK?
And the way it goes
is that you have
to get your vector ready to
receive your gene of interest.
Usually, you cut your vector
with the same restriction
enzyme or enzymes that you've
cut your gene of interest with.
And then the ends can go
together in a matching way.
And you'll get a covalently
closed recombinant DNA
molecule that has
got both your vector
and your gene of interest.
So you're going to
prepare a vector.
And you'll cut it with
a restriction enzyme
or restriction enzymes
the same as used
to cut the gene of interest.
You then mix together
your cut vector
and your cut gene of interest.
And they should have, at
this point, compatible ends.
That is your job as
a genetic engineer,
to make sure that that's true.
You add DNA ligase to get
bonding and a single vector
gene of interest molecule.
And then you take the mix.
You usually have millions
and millions of molecules
that you're doing this with.
And you take the whole mix.
And you insert it into
the host bacterium, OK?
You insert-- and the
correct term is transform.
You insert or transform the
whole mix into host bacteria.
And these are the bacteria that
that vector likes to grow in.
And the last step in this, then,
is that not all the bacteria
are going to get one
of these molecules,
these recombinant DNA
molecules inserted into them.
In fact, most won't.
It's only a tiny fraction
of a percentage that
are going to get a
molecule of vector
plus gene of interest inserted.
So you want to get rid
of all the bacteria
that don't have your
recombinant clone.
And this is where this
selectable marker we mentioned
comes in.
You select for bacteria
that have got the vector
gene of interest construct.
And usually, the selection
is with a particular type
of chemical that
is an antibiotic.
And so you put an
antibiotic resistance gene
into the plasmid.
And that allows
the bacteria that
have got the vector plus
the gene of interest
to grow in the presence
of antibiotics.
But don't worry
about that for now.
But you select for bacteria
and the gene of interest.
And then this allows the
vector GOI to specifically grow
in the bacteria.
And you can then go ahead
and isolate and use the DNA
from this growth mix.
I've drawn this for
you in this next slide,
where you can see a
gene of interest is cut.
The plasmid vector with its
origin of replication is cut.
The two DNAs are ligated.
They've got compatible ends.
And you can actually get three
outcomes of this ligation.
You might get your gene of
interest just circularizing.
That's not going to
be very productive.
Those DNAs will just disappear
later in the process.
You might get the vector
only that ligates.
That can be a bit of a problem.
And there are ways you
can get rid of those.
And then you might get
the correct construct,
which is your gene of
interest plus the vector.
And that's what you want.
You take that ligation mix,
and transform bacteria with it,
and grow, as we said, a lot
of the recombinant clone.
The selectable marker
comes in because you
can grow your transformants--
the vector or the vector
plus gene of interest--
on, in this case, ampicillin,
which is an antibiotic.
Normally, ampicillin will
stop bacteria from growing.
That's why, if you get
a bacterial infection,
antibiotics are the things
that you take to treat it.
You can use that.
The bacteria-- we got
antibiotics from bacteria.
And now, we can use them
in molecular cloning, OK?
So here, there is an
ampicillin resistance gene also
on the plasmid vector.
If you grow the bacteria in
the presence of ampicillin,
only the bacteria that took
up the vector with ampicillin
resistance will be
able to grow, OK?
So that's a way to get rid of
all the other non-transformed
bacterial clones.
Good.
You've got a lot
of information now
about how you put together
vectors and inserts, or genes
of interest.
And part of it is the
notion of compatible ends.
So I want you to do some
practice in this class
exercise, and see if you
are able to use the concepts
in a practical way.
