PROFESSOR: Hello, and welcome
to another help session on
recombinant DNA.
Today we're going to
discuss genomic
libraries and cDNA libraries.
So what is a genomic library?
Well, with most organisms
the genome is really too
large to deal with.
It's too large to work
with or to examine.
So oftentimes it's more useful
to cut it down into smaller
pieces that you can
then examine.
How do we make a genomic
library?
Well, we start off with
our cell of interest.
And the first thing that we're
going to do is we're going to
isolate all the DNA
from the cell.
Once we have the DNA, we're
going to use restriction
enzymes to cut it into
smaller sections.
Now, we have to be very careful
when we choose which
restriction enzyme
we want to use.
We want to use an enzyme that
has enough restriction sites
in the genome so that we'll get
small enough pieces to put
in the vector, but not
so small that all the
genes get cut up.
Usually what we we'll use for
the vector is a plasmid.
A plasmid is a very standard
and easy way to get foreign
DNA into a bacteria.
So we're going to cut up our DNA
and the plasmid with our
restriction enzyme.
Sometimes we might want to
even make two different
libraries using two different
restriction enzymes so that
we'll have the cuts in
different places.
Once we have our cut up DNA and
our plasmids, now we need
to ligate them together
using DNA ligase.
Once the cDNA is pasted into the
plasmid, now we're ready
to actually transform
them into bacteria.
It's very easy to transform
plasmids into bacteria.
There are multiple different
ways to do it.
One common one is to use heat
shock, whereby you heat the
bacteria up and then cool it
down rapidly, which causes
little holes to open
up that allows the
plasmids to be taken up.
What we end up with are
thousands of different
bacteria cells, all with
different sections of the DNA.
So combined, all these different
sections form the
genome, the original genome that
we have, but they're in
much smaller sections.
They're much easier
to work with.
And this combination of all
these different cells forms
our genomic library.
So that's one type of library.
Another type of library
is a cDNA library.
So the way that the cDNA library
is different from a
genomic library is that while
the genomic library
encompasses the entire genome of
the cell, the cDNA library
just looks at what genes are
being expressed in the cell.
So let's go over how we would
make a cDNA library.
What we're going to start off
with is we're going to start
off by isolating the mRNA.
So the mRNA, clearly, is
the RNA that's being
expressed in the cell.
This is what's ultimately-- the
proteins are going to be
produced by the cell.
We're then going to use reverse
transcription to
create the DNA version
of the RNA.
This is what we're referring
to, again, as the cDNA.
Once we have the cDNA, we then
need to add restriction sites
to the ends of them.
Remember, before we were just
able to use innate restriction
sites in the genomes.
But for the cDNA library,
we really want to
keep the cDNA intact.
We don't want to cut it up.
So we just want to add
restriction sites to the ends
of the cDNA.
Then we can treat the cDNA
and the plasmids
with restriction enzymes.
Once again, once they've been
cut up we can use DNA ligase
to combine the cut up plasmids
and the cut up cDNA to the
complete plasmid plus cDNA.
And finally, once again, we can
transform our bacteria,
and then the combination of
all these cells, all with
different types of cDNA,
form our cDNA library.
And cDNA libraries are very
useful if you want to compare
the difference in protein
expressions between two
different cells.
Thank you very much.
This has been another help
session for recombinant DNA.
