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PROFESSOR ROBERT DORKIN:Hi, and
welcome to a help session

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on recombinant DNA.

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Today we will be talking about
the polymerase chain reaction

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as well as DNA sequencing.

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The polymerase chain reaction,
also known as

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PCR, has many uses.

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One of the most common
uses is to amplify a

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desired section of DNA.

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What you need for the reaction
is your DNA sequence of

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interest, DNA polymerase, DNA
primers, and then four

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different nucleotides.

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You combine all these together,
and the first thing

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that you do is you heat
the reaction up.

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What this does is that by adding
heat to the system, you

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break the hydrogen bonds
between the two

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different DNA strands.

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This results in two separate
DNA sequences.

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Now what happens is that you
allow the system to cool.

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As it cools, the DNA primers are
able to hybridize to this

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separate strands.

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Now as you remember from
lecture, when you're

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synthesizing DNA, you synthesize
from the five prime

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to three prime direction.

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This means that the primers you
design have to match the

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three prime end of your
sequences of interest.

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So for example, if we were
designing primers for these

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two sequences, one of them would
be GGTA and the other

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one would be AGCT.

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Now, I've written four here.

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In actuality, these primers are
generally longer, around

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16 or so, 16 to 27.

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However, they can be a whole
different variety of lengths

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dependent on numerous
different factors.

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The next thing that happens is
that the DNA polymerase is

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going to bind to the DNA
sequence with the primer.

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Once the DNA polymerase is
bound, it's going to take some

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of the free nucleotides in the
surrounding area and slowly

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add them to finish
up the strand.

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And so on and so forth.

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Once it's completed, we are
going to have now doubled our

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original DNA sequence.

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We're going to have two strands
that are identical to

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the first one.

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As you can see, by repeating
the steps, heating it,

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allowing it to cool, allowing
more primers to bond, and then

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allowing the DNA polymerase to
elongate, we can double the

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number of sequences
every round.

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And you can rapidly
get a large amount

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of the desired sequence.

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PCR has other uses though
besides simply increasing the

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total amount of DNA
that you have.

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One of the uses of PCR
is to sequence DNA.

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Now, if we look over here,
normally DNA is form of

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deoxyribonucleic acids.

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You have the phosphate group
on the five prime end.

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You have a hydroxyl group
on the three prime end.

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This hydroxyl group
is very important.

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That's because when a new
nucleotide is added, this

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hydroxyl group undergoes a
covalent bond with the

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phosphate on the new nucleotide
and then adds a new

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nucleotide that way.

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So you can see you're adding
the five prime

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the three prime direction.

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However, it is possible
to create a

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dideoxyribonucleic acid.

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The dideoxyribonucleic acid,
instead of having a three

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prime hydroxyl group, has
a three prime hydrogen.

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This three prime hydrogen is no
longer capable of forming a

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covalent bond with
a phosphate.

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That means as soon as the
dideoxyribonucleic acid is

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added to DNA, no further
nucleotides can be added in

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the series.

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Let's go back to our example
with the primers.

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What does that mean for here?

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Well, let's say you have a
normal PCR reaction, but in

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addition to the four
deoxyribonucleic acids you

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have, you also take a little
bit of dideoxyribonucleic

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acids of one of the types.

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So let's say we add
in some ddTTP.

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Now what happens is that your
DNA polymerase will go along

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adding nucleotides as normal,
but if it ever adds a

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dideoxyribonucleic acid, the
polymerase will stop.

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So if it adds, say
a normal T here--

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continues down, continues
down.

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If it adds a dideoxyribonucleic
acid here,

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it's going to stop,
and we're going to

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get a truncated sequence.

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And so you can see that at any
position that we have an A,

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it's going to be possible to
have a truncated sequence of

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that length.

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What this means that we're now
going to, once the PCR is

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complete, have different DNA
sequences of numerous

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different lengths.

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But the one thing they're all
going to have in common is

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that they're all going to end
with a T. So you can imagine

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doing this now for each of the
four different letters.

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Then we can take them and
run them out of a gel.

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Let's go look at such
a gel over here.

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Here we have a gel.

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Each of these letters
represents which

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dideoxyribonucleic acid was
used for that experiment.

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And then the PCR was
run out on the gel.

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As you remember, the strands
close to the bottom are the

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shorter strands, and the strands
close to the top are

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the longer strands.

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So if we look at this gel, we
know that the shortest strand

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ends with a G. The next shortest
strand ends in a T.

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Oh, sorry, ends in
an A, excuse me.

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Then the next short strand ends
in a T, then two A's,

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then a G, then a C, then a T. So
as you can see, this is one

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way to determine the
sequence of DNA.

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Another way has been devised,
which is even faster.

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Instead of running the sequences
all out in different

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polymerase chain reactions, what
they do is they have some

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of each of the
dideoxynucleotides together.

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But now, they fluorescently
label them, such that you have

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a different fluorescent
label on each

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dideoxyribonucleic acid.

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Now what happens is that
you can run it

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out all on one column.

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And then by just looking at the
colors, you can determine

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what the sequence is.

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So once again, the sequence
would be GATAAGCT.

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And so this way, you can more
efficiently, more rapidly,

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determine what the
DNA sequence is.

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This has been two examples of
polymerase chain reactions and

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their uses.

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This has been another help
session on recombinant DNA.

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We hope you join us
again next time.

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Thank you.