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.