PROFESSOR ROBERT DORKIN:Hi, and
welcome to a help session
on recombinant DNA.
Today we will be talking about
the polymerase chain reaction
as well as DNA sequencing.
The polymerase chain reaction,
also known as
PCR, has many uses.
One of the most common
uses is to amplify a
desired section of DNA.
What you need for the reaction
is your DNA sequence of
interest, DNA polymerase, DNA
primers, and then four
different nucleotides.
You combine all these together,
and the first thing
that you do is you heat
the reaction up.
What this does is that by adding
heat to the system, you
break the hydrogen bonds
between the two
different DNA strands.
This results in two separate
DNA sequences.
Now what happens is that you
allow the system to cool.
As it cools, the DNA primers are
able to hybridize to this
separate strands.
Now as you remember from
lecture, when you're
synthesizing DNA, you synthesize
from the five prime
to three prime direction.
This means that the primers you
design have to match the
three prime end of your
sequences of interest.
So for example, if we were
designing primers for these
two sequences, one of them would
be GGTA and the other
one would be AGCT.
Now, I've written four here.
In actuality, these primers are
generally longer, around
16 or so, 16 to 27.
However, they can be a whole
different variety of lengths
dependent on numerous
different factors.
The next thing that happens is
that the DNA polymerase is
going to bind to the DNA
sequence with the primer.
Once the DNA polymerase is
bound, it's going to take some
of the free nucleotides in the
surrounding area and slowly
add them to finish
up the strand.
And so on and so forth.
Once it's completed, we are
going to have now doubled our
original DNA sequence.
We're going to have two strands
that are identical to
the first one.
As you can see, by repeating
the steps, heating it,
allowing it to cool, allowing
more primers to bond, and then
allowing the DNA polymerase to
elongate, we can double the
number of sequences
every round.
And you can rapidly
get a large amount
of the desired sequence.
PCR has other uses though
besides simply increasing the
total amount of DNA
that you have.
One of the uses of PCR
is to sequence DNA.
Now, if we look over here,
normally DNA is form of
deoxyribonucleic acids.
You have the phosphate group
on the five prime end.
You have a hydroxyl group
on the three prime end.
This hydroxyl group
is very important.
That's because when a new
nucleotide is added, this
hydroxyl group undergoes a
covalent bond with the
phosphate on the new nucleotide
and then adds a new
nucleotide that way.
So you can see you're adding
the five prime
the three prime direction.
However, it is possible
to create a
dideoxyribonucleic acid.
The dideoxyribonucleic acid,
instead of having a three
prime hydroxyl group, has
a three prime hydrogen.
This three prime hydrogen is no
longer capable of forming a
covalent bond with
a phosphate.
That means as soon as the
dideoxyribonucleic acid is
added to DNA, no further
nucleotides can be added in
the series.
Let's go back to our example
with the primers.
What does that mean for here?
Well, let's say you have a
normal PCR reaction, but in
addition to the four
deoxyribonucleic acids you
have, you also take a little
bit of dideoxyribonucleic
acids of one of the types.
So let's say we add
in some ddTTP.
Now what happens is that your
DNA polymerase will go along
adding nucleotides as normal,
but if it ever adds a
dideoxyribonucleic acid, the
polymerase will stop.
So if it adds, say
a normal T here--
continues down, continues
down.
If it adds a dideoxyribonucleic
acid here,
it's going to stop,
and we're going to
get a truncated sequence.
And so you can see that at any
position that we have an A,
it's going to be possible to
have a truncated sequence of
that length.
What this means that we're now
going to, once the PCR is
complete, have different DNA
sequences of numerous
different lengths.
But the one thing they're all
going to have in common is
that they're all going to end
with a T. So you can imagine
doing this now for each of the
four different letters.
Then we can take them and
run them out of a gel.
Let's go look at such
a gel over here.
Here we have a gel.
Each of these letters
represents which
dideoxyribonucleic acid was
used for that experiment.
And then the PCR was
run out on the gel.
As you remember, the strands
close to the bottom are the
shorter strands, and the strands
close to the top are
the longer strands.
So if we look at this gel, we
know that the shortest strand
ends with a G. The next shortest
strand ends in a T.
Oh, sorry, ends in
an A, excuse me.
Then the next short strand ends
in a T, then two A's,
then a G, then a C, then a T. So
as you can see, this is one
way to determine the
sequence of DNA.
Another way has been devised,
which is even faster.
Instead of running the sequences
all out in different
polymerase chain reactions, what
they do is they have some
of each of the
dideoxynucleotides together.
But now, they fluorescently
label them, such that you have
a different fluorescent
label on each
dideoxyribonucleic acid.
Now what happens is that
you can run it
out all on one column.
And then by just looking at the
colors, you can determine
what the sequence is.
So once again, the sequence
would be GATAAGCT.
And so this way, you can more
efficiently, more rapidly,
determine what the
DNA sequence is.
This has been two examples of
polymerase chain reactions and
their uses.
This has been another help
session on recombinant DNA.
We hope you join us
again next time.
Thank you.
