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Good morning.

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It can't go without at least
some acknowledgement dimension.

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If you should ever
find yourself in

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life in a situation where you have
or are about to give up all hope,

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you think things are utterly
impossible and there's no way,

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you will remember this week
that nothing is impossible.

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It is possible to come back three
games down in the bottom of the

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ninth inning, you've got
to believe you can do it,

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and remember to have
Dave Roberts pinch run.

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Just a general bit of good
advice. What an amazing week, just

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absolutely amazing week. Wow.
There are lessons in life to

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be taken from it.
Please do take them.

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You know, there really are. I
mean I'd given up hope by that

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point, I confess. I
wish I could say oh,

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I knew they were going to
pull it out, but I didn't.

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And, boy, they pulled it
out one game at a time.

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So, all of you think good thoughts
this week. This could be a historic

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week, you know, you were
here. Anyway, onward.

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We were talking
last time about how

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to analyze your clone. The
notion of cloning random pieces

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of DNA, identifying your
clone within a library,

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purifying the DNA from a clone,
doing some preliminary analysis by

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maybe cutting with a restriction
enzyme, then sequencing it using

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these techniques that I'd described
would allow you to take the clone

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that was, say, able to
rescue the yeast that

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couldn't grow without arginine
and figure out what its

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DNA sequence was. You could
take the clone that you

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had obtained by hybridizing with
the DNA sequence corresponding to the

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protein sequence for beta-globin and
sequence it and see the beta-globin

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gene sequence perhaps.
This is very powerful.

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I want to take a brief moment,
we'll come back to it in more detail

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in a subsequent lecture, but I
really described how you would

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sequence one clone. I
just want to make a note,

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because someone asked about it last
time, about how you would sequence

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an entire genome.
Someone asked about this.

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Remember before we pulled out
our clone, we sequenced it,

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we got its DNA sequence. What
if I wanted to sequence the

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entirety of a genome?
Yeah. Do a lot of this,

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right, basically if I
got a whole genome. Well,

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somebody asked could I put a
primer here and just sequence?

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It would take a very long time.
And it turns out that it wouldn't

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work because the separation that
you can achieve through gels is a

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function, the separation between N
and N plus 1 in length goes like the

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logarithm of the ratio. So,
it turns out that when N and N

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plus 1 get to like about a thousand,
you can achieve very little physical

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separation between them. And
so, DNA sequencing runs cannot

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go much past the thousand bases.
So, the problem with sequencing a

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genome by putting down a primer on
an extraordinarily long piece of DNA,

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a hundred million bases, is
you cannot separate the little

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fragments like that. So,
what you do is you break up

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your genome into lots of pieces.
One strategy, break it up into a

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library of some very big pieces.
It turns out you can make pieces at

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random of a hundred
thousand base pairs.

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Cloning these in bacterial
artificial chromosomes,

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as we talked about before.
Take a library of bacterial

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artificial chromosomes and
then begin sequencing them.

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And take any given bacterial
artificial chromosome and break it

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up into a whole lot of pieces that
are maybe a thousand bases long,

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and you could sequence all of those.
How do you arrange to get just a

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perfect overlapping set of thousand
based pair clones that perfectly

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tile across the sequence
with no redundancy?

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You don't. That's the correct
answer. That's how you do it,

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you don't. Instead you just
randomly take a bunch of things.

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And, in fact, typically you might
take clones that give you six or

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eight-fold redundancy. You
just sequence a lot of clones

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and then you ask the
computer to reassemble it.

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And, in fact, all that overlap is
very good for being able to stick

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these pieces together. Sometimes
people do such things as

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take pieces that might be four
thousand bases long and sequence a

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thousand bases here and a thousand
bases here by using a primer that

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starts there and a primer that
starts there. And then you can get

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DNA sequences from two ends of
a clone. And if you had that for

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zillions of clones your computer
program might do an even better job

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of linking things up. It's
one very big crossword puzzle

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of putting together all of these
pieces, a jigsaw puzzle of putting

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together all these pieces.
But, in effect, this is how you

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sequence a big piece of DNA.
You chop it up into medium-sized

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pieces of DNA and then tinny
pieces of DNA, you sequence them,

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and you use computational
science to reassemble it.

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Some people, for some genomes,
take the whole big genome and

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immediately go to lots of
little pieces. That can work,

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too. I depends on exactly how
complicated your genome is.

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In the human genome, there are some
parts of a human genome that are

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almost identical that might be like
99.91% identical in two different

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parts of the genome.
And so, if you do that,

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you may have trouble telling
those pieces apart. So,

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for really complicated genomes
people like sometimes breaking it up

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into intermediate-sized pieces.
But basically the idea of

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sequencing a big piece of DNA
by this process is referred to as

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shotgun sequencing.
Shotgun sequencing,

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in fact, was developed in
about 1980 by Fred Sanger,

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the same guy who developed the DNA
sequencing technique that I told you

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about using polymerase
and dideoxynucleotides.

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Sanger very quickly wanted to go
from sequencing a single piece to

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sequencing pieces, and so
he developed the shotgun

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technique there. And it's
now been applied in many

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different forms of intermediate
shotguns, whole genome shotguns,

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et cetera. So, that's in reply to
the question someone asked last time

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about, well, how would
you do a whole genome? And,

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as a matter of fact, this
is not theoretical because,

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in fact, people do hold genomes
this way. And we do this at MIT.

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Lots of genomes get done here in
this fashion. Someone else asked

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how would you analyze your clone.
And, again, I'll just make a brief

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remark on that in response
to the question. So,

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analyzing some DNA sequence.
So, suppose we got some DNA

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sequence, A-A-T-A, don't
bother writing this down.

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I'm just making up letters here.
How would we make any sense of it?

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Suppose I give you the ones
and zeros from your hard-drive,

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how would you make any sense out of
them? This is about as interesting

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as the ones as yours from
your hard-drive, right?

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It's got four letters, not
two, but this is actually what

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you get out of any project. You
want to sequence beta-globin?

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You'll get something like this.
You want to sequence the arginine

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gene? You want to sequence the
human genome? You get a very long

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string of four letters.
What do you do with it?

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Oh, well, you could compare it
to a normal copy of the gene.

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And if I did that I might
find a bunch of differences.

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But how would I even know where
the beta-globin gene was within this

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sequence? This clone contains
beta-globin. How would I even find

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the exons? Yes? Or
whatever? Look at codons.

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So, let's start looking at
the codons. This codon here?

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Well, or this, maybe it's
this codon here. Sorry?

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Find it. Do you see any
start codons here? Oh,

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there's an ATG there. So,
maybe that's the start codon or

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maybe not. How often do we
expect to find an ATG in some

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reading frame? You know,
it could happen fairly

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easily. Also, how do we
know it's going this way?

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Maybe we should look for
an ATG, we'll put it there,

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going this way. Sorry?
I drew the arrow there?

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Well, that's because it's where
the sequence started out on my page.

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It doesn't tell me my gene
runs that way. Yes? From five

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prime to three prime. Ah, but
it's a double-stranded piece

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of DNA. You see, if it's
five prime to three prime on

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this strand, the genome has a,
another strand that reads the other

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way. What did I get?
C-A-T-A, right, C-C-T,

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et cetera. And the gene could
be encoded on this strand.

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This could be the coding strand,
that could be the coding strand, and

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looking for a mere ATG in one of
three possible reading frames on one

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of two possible strands
I'll find all sorts of stuff.

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So, sorry? Guess.
Guess. Guess is good.

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They don't, don't, won't you, you
remember we talked about getting

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papers accepted. If you
were to write up the paper

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that way the reviewing would
probably ding it and say,

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you know, the guess isn't,
isn't good enough. So, that's

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actually very interesting. How
do you actually find the gene

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sequence? Well, it
turns out to be a

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non-trivial problem which often
gets glossed over in the textbooks.

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What you might do is if
something really were exonic,

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if this were any exon, does
it have any properties that you

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can think of? It shouldn't have
a stop codon. No stop codon.

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How often does a stop codon occur
at random in a given reading frame?

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How many stop codons are there?
Three out of 64 possible codons.

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There's about one in 20 codons in
any given reading frame is a stop

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codon. So, that means if
I read for about 20 codons,

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and I don't encounter a stop,
it's beginning to get more likely

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that that's not random. If
I read for, say, 60 codons,

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180 bases and I've encountered no
stop codon in that reading frame,

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that chances of that occurring is
about either the minus three or so,

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right? Because if I went through
three characteristic lengths,

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either the minus three, you know,
and I don't know, about 5% or

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something like that. If I
went for thousands of bases

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without any stop codon,
would you be impressed? That's

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pretty impressive. So, all
I have to do is find the

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few thousands of basis with no
stop codon. The problem with that is

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that in bacteria there are some
genes that are a thousand bases long

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and you, there, you can
read them and they have no

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stop codon. What's the
problem with the human

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genome? Introns. It
turns out that because the

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coding sequences are broken up into
small exons, if I found a thousand

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bases with no stop codons then
it's very likely coding sequence.

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But a typical human exon is on
the order of a 150 to 200 bases.

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Very inconvenient because,
you know, it's a typical exon

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encodes 50, 60, 70 codons.
So, it turns out that

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even that is not so easy to do.
Well, the answer is it's not a

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trivial problem. People
do all sorts of things to

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figure out how to decode sequences
of genomes. You do run computer

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filters across there that
say, look, there are a bunch of

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consecutive codons
without stop codons.

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There tend to be little preferences,
like amongst the synonymous choices

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of stop codons, humans tend
to prefer one stop codon,

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one codon for a specific
amino acid over others. So,

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there are some biases as
to which codons get used.

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And the computer can kind of take
a little bit of account of that.

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Then you can also have made a
library of seed DNAs and sequence

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seed DNA, the mRNA which will help
you a lot and look for where they

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match up. Then you can take
sequences from the human

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and the mouse. And it
turns out that the sequences

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in the mouse and the sequences
in the human, if you line them up,

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the exons tend to match up better
than the introns because evolution

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cares a lot about the exons.
But it turns out this is not a

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trivial problem. And even
today, if I give you a

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random stretch of human DNA,
it's not, there is no simple

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computer program that it's
on, that on its own, not even a

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complicated computer program,
but on its own would be able,

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without axillary data, to
accurately pick out all the genes.

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Even for simple bacteria, we
cannot nail perfectly all the

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genes. Although, the lack
of these introns means that

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the exons tend to be pretty big,
it means the coding are pretty big

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and we can kind of do it. So,
I just wanted to point out that,

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that there's a lot still to be
done there. The cell manages,

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thank you, to read this just fine,
but we're not as smart yet as the

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cell, and so we're not totally
able to read out all this stuff.

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We'll come back to genomics in a,
in a further lecture. Yes? Yeah,

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wouldn't --
What a cool idea.

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Yes. There are actually some
experiments, which maybe if you

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remind me we can, I can
work it into a subsequent

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lecture, but people have some
experiments where they can randomly

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mutagenize zillions of bacteria and
determine which ones will grow and

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which ones won't. And they
can do it all in parallel

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in a single test-tube. And
thereby you can tell which,

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which nucleotides in the
genome matter and which don't.

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It's a kind of cool procedure.
OK. Anyway, I just wanted to sort

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of tie up that bit here. Now,
let's move on to re-sequencing

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a gene. So, let's
suppose we've managed to

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sequence, I don't know, the
human genome, the entirety of

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the human genome we have before
us, OK? Actually, next week in the

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journal Nature will appear
a paper reporting, in fact,

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today, yester, no, yesterday, in
fact, it was yesterday, yesterday

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appeared in the journal Nature
a paper reporting the finished

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sequence of the
human genome.

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http://www.nature. om/nature/journal/v431/n7011/pdf/nature03001.pdf

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And so, anybody
who wants to go

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online, in fact, we can
get, we'll get copies for the

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class. Why don't we get you copies
of the paper? It's not as long as

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the last one. But, in fact,
I didn't realize it was

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yesterday. I thought it was next
week. Yesterday came out the final

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report on the finished
sequence of the human genome,

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which a number of us have been
laboring on for quite a long time.

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And it just appeared. So,
we actually have that now.

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It actually, it's been on the
Web for a while, but the paper

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describing it took a while to
write up and it came out yesterday.

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So, we'll get you a copy of that.
But now you've got that whole

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sequence of the human genome here.
I've been, you know, I've been

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working on this paper with
people for so long that,

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you know, I hadn't actually paid
attention to the fact that it just

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came out. You don't want to know
how long it took to write this.

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The paper, actually, is unusual.
It's the only paper that Nature has

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ever published where the author list
is sufficiently long that we don't

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have it in the Journal. There's
a website that contains the

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author list. There are, I
believe, I don't have the final

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count, but something in the
neighborhood of about 200 authors to

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the paper. We decided that
everybody who'd worked on it should

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be a co-author of the paper, and
we just put it all on a website.

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So, anyway, I digress. So, suppose
we have the beta-globin gene here.

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So, I've got that in -- I've
got the normal form of the

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beta-globin gene, or I've
got one person's form,

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in any case, in the human genome
sequence. Now I want to take a

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patient with sickle cell anemia and
I want to re-sequence their gene.

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Now, remember what we said, we
would, we would make a library from

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that person, right? So,
we'd get that person's blood,

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we'd purify DNA, we'd cut it, we'd
clone it, we'd probe the library

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with a radioactive probe
for the beta-globin gene,

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we'd pull out the gene
and we'd re-sequence it.

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Suppose we wanted to do
that to a hundred patients.

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For every patient we'd get blood,
we'd make DNA, we'd clone in a

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plasma, we'd made a whole plasma
library, plate it out on filter,

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probe it with a radioactive probe,
pull out the clone and sequence it.

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Now, for any such library you
probably need to look through a

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couple hundred thousand
clones to find beta-globin.

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So, for your DNA and your
DNA and your DNA and your DNA,

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we're going to make libraries
of a hundred thousand clones,

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that's a couple, that's
a lot of plates, right?

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00:18:29,000 --> 00:18:32,000
We're going to put them all
on, on nylon filters in these

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Seal-a-Meal bags with
these radioactive probes,

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and we're going to look
for your beta-globin clone,

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your beta-globin clone,
your beta-globin clone, your

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beta-globin clone,
et cetera, et cetera,

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et cetera. This is really boring.
Do you realize how off putting it

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would be to study sickle cell
anemia if we had to do that for each

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successive patient,
make a whole library?

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But that was what you had to do in
molecular biology because that was

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how you got the gene.
You build a whole library,

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you withdraw it from the library.
However, if you wanted to do this,

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could you manage to get the
beta-globin sequence from your

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genome without having to
make the whole library?

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It turns out, and I know it's been
covered at least in some of the

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sections, there's a cool
technique to do that. And what is

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that technique? PCR. So,
it turns out that the next

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00:19:32,000 --> 00:19:39,000
really great advance in molecular
biology was the technique of PCR.

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00:19:39,000 --> 00:19:47,000
And what PCR was a way, is, is
a way to obtain a piece of DNA

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corresponding to an already known
gene, you have to already know the

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gene, and what it allows you to
do is then obtain that piece of DNA

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based on knowing at least
some of its sequence.

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It allows you to amplify just that
DNA from a, from any individual.

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So, as compared to the experiment
where I make a library for you and a

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00:20:20,000 --> 00:20:25,000
library for you and a library
from you and a library from you,

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00:20:25,000 --> 00:20:31,000
each of which could take a month,
PCR would allow us to do it in

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00:20:31,000 --> 00:20:36,000
principle in five
minutes. And, actually,

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00:20:36,000 --> 00:20:41,000
there are machines that would
let you do it in five minutes.

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So, let's discuss how this PCR
works. Nobody uses the five minute

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00:20:46,000 --> 00:20:52,000
machines because you usually
will then wait an hour or so,

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00:20:52,000 --> 00:20:57,000
but anyway. Suppose I take
my DNA sequence here from

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00:20:57,000 --> 00:21:04,000
the human genome. Five
prime to three prime.

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Five prime to three prime.
This sequence here beta-globin.

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00:21:14,000 --> 00:21:23,000
I want to obtain that sequence.
The first thing I do is I'm going to

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heat my DNA sample to maybe
97 degrees Celsius to denature.

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00:21:33,000 --> 00:21:42,000
Denaturing means, of
course, breaking the hydrogen

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00:21:42,000 --> 00:21:51,000
bonds that separate the two strands
so that the strands come apart,

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00:21:51,000 --> 00:22:00,000
five prime to three prime,
five prime to three prime.

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00:22:00,000 --> 00:22:09,000
Now, what I then do is I take a
specific DNA primer matching this

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00:22:09,000 --> 00:22:19,000
stretch just before the
beta-globin gene starts.

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00:22:19,000 --> 00:22:22,000
Or just before where I'm interested
in. How do I make a primer that

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00:22:22,000 --> 00:22:26,000
matches just that sequence? I
order, well, how do I know what

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00:22:26,000 --> 00:22:30,000
to order? I know
the sequence, right?

305
00:22:30,000 --> 00:22:34,000
I've got the sequence already. I
just look at it and I say I want

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00:22:34,000 --> 00:22:38,000
that sequence. And
then how do I get it?

307
00:22:38,000 --> 00:22:42,000
I order it. I type it into the Web
and the machine will synthesize me

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00:22:42,000 --> 00:22:47,000
this, this primer. Typically
a 20-base stretch will

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00:22:47,000 --> 00:22:51,000
suffice. So, I'll get me a
twentymer, a 20 base oligonucleotide

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00:22:51,000 --> 00:22:55,000
complimentary to the sequence
on this side of the gene.

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00:22:55,000 --> 00:23:00,000
What I'm also going to do
is the same thing over here.

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00:23:00,000 --> 00:23:06,000
I'm going to get a second primer.
This is primer number one. This is

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00:23:06,000 --> 00:23:12,000
primer number two.
OK? Now, let's see.

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00:23:12,000 --> 00:23:19,000
Five prime. This is five prime,
five prime. Now what I'd like to do

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00:23:19,000 --> 00:23:25,000
is add polymerase,
I'd like to add dNTPs.

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00:23:25,000 --> 00:23:32,000
So, plus DNA
polymerase plus dNTPs.

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00:23:32,000 --> 00:23:36,000
And what will happen?
Polymerase will come along and

318
00:23:36,000 --> 00:23:41,000
start copying my DNA, but
it will only copy it starting

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00:23:41,000 --> 00:23:45,000
from the primers. Now,
this will keep going,

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00:23:45,000 --> 00:23:50,000
of course, but DNA
polymerase doesn't go forever,

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00:23:50,000 --> 00:23:55,000
you know, the reactions
sort of stops at some point.

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00:23:55,000 --> 00:23:59,000
And so you'll get a strand
going off here and a strand

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00:23:59,000 --> 00:24:04,000
going off there. Now,
notice what I've done.

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00:24:04,000 --> 00:24:10,000
I started with an entire human
genome, and the number of copies of

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00:24:10,000 --> 00:24:15,000
beta-globin was one per genome.
When I'm done with this process,

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00:24:15,000 --> 00:24:21,000
how many copies, how many
double-stranded copies of

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00:24:21,000 --> 00:24:27,000
beta-globin do I have? Two.
That's still very little,

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00:24:27,000 --> 00:24:33,000
but it's more than I had
before. So, what do I do next?

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00:24:33,000 --> 00:24:41,000
Repeat. So, let's heat up that
sample again. We'll denature at 97

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00:24:41,000 --> 00:24:48,000
degrees, and now we have our initial
strand here, we have our strand that

331
00:24:48,000 --> 00:24:56,000
came off this primer that runs
to here and maybe goes forward,

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00:24:56,000 --> 00:25:05,000
we have this strand here.
We have this strand here.

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00:25:05,000 --> 00:25:15,000
And this was five prime, five
prime, five prime, five prime.

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00:25:15,000 --> 00:25:25,000
Now what do we do? We repeat.
We'll take our primer, this is

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00:25:25,000 --> 00:25:33,000
primer number one, let's
see. It matches over there.

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00:25:33,000 --> 00:25:41,000
Primer number two over here.
Number one over here. Number two.

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00:25:41,000 --> 00:25:48,000
Have I got this right? Yes. Good.
Then where does this guy stop?

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00:25:48,000 --> 00:25:56,000
Right at the end where my
other primer was. This guy

339
00:25:56,000 --> 00:26:02,000
runs along here. That guy
stops right at the end.

340
00:26:02,000 --> 00:26:07,000
That guy might go a little further.
How many copies of the beta-globin

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00:26:07,000 --> 00:26:12,000
gene do I have now?
Four. Two of which,

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00:26:12,000 --> 00:26:17,000
by the way, perfectly sit between
my pink primers. What's going to

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00:26:17,000 --> 00:26:22,000
happen if I do this again?
How many copies will I get?

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00:26:22,000 --> 00:26:27,000
Eight, six of which will sit
perfectly and two might be a little

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00:26:27,000 --> 00:26:32,000
ragged as to where
they go. So, initially,

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00:26:32,000 --> 00:26:38,000
after cycle number zero,
that is initial conditions,

347
00:26:38,000 --> 00:26:44,000
the number of copies relative
to the genome was one.

348
00:26:44,000 --> 00:26:50,000
After one cycle it's two.
After two cycles it's four.

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00:26:50,000 --> 00:26:56,000
After N cycles it's two to
the N copies. Is that clear

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00:26:56,000 --> 00:27:01,000
how the PCR works? And
that on every round you're

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00:27:01,000 --> 00:27:07,000
doubling. And, with the
exception of those two

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00:27:07,000 --> 00:27:13,000
white things that go off to the
side, they're going back and forth and

353
00:27:13,000 --> 00:27:18,000
back and forth between the two
primers you chose to put in.

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00:27:18,000 --> 00:27:24,000
What is when N equals ten, what
do you got? A thousand copies.

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00:27:24,000 --> 00:27:30,000
What happens when N equals
20? A million copies.

356
00:27:30,000 --> 00:27:34,000
What is the copy number
of beta-globin? Beta,

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00:27:34,000 --> 00:27:38,000
let's suppose beta-globin,
for the sake of the argument,

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00:27:38,000 --> 00:27:42,000
sake of argument is
about one thousand bases.

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00:27:42,000 --> 00:27:46,000
What fraction of the human
genome does beta-globin represent?

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00:27:46,000 --> 00:27:50,000
Yeah, about a millionth for
the genome. No, actually,

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00:27:50,000 --> 00:27:54,000
one three millionth, but
we'll call it a millionth.

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00:27:54,000 --> 00:27:58,000
So, after I've made a million-fold
amplification of beta-globin,

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00:27:58,000 --> 00:28:03,000
beta-globin now represents half
of the stuff that's in the tube.

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00:28:03,000 --> 00:28:08,000
What would happen if I go another
ten rounds? How many copies do I

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00:28:08,000 --> 00:28:13,000
have? A billion copies. So,
in other words, I started with

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00:28:13,000 --> 00:28:18,000
something that was only present at
about one one-millionth of what was

367
00:28:18,000 --> 00:28:23,000
in my test tube. If I could
make a billion copies of

368
00:28:23,000 --> 00:28:28,000
that specific molecule, now it
so dominates the mixture that

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00:28:28,000 --> 00:28:33,000
it is a thousand times more
abundant than the rest of the genome.

370
00:28:33,000 --> 00:28:39,000
It works. That's the
remarkable thing, this works.

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00:28:39,000 --> 00:28:45,000
Any questions about the technique?
Now, yes? Well, I need two primers

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00:28:45,000 --> 00:28:52,000
in their sequence. How many
copies do I need of each

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00:28:52,000 --> 00:28:58,000
of those primers? Well, I,
I obviously need a lot of

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00:28:58,000 --> 00:29:03,000
copies of those primers.
So, primer number one,

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00:29:03,000 --> 00:29:06,000
it's a single sequence, but
when I order it from the company,

376
00:29:06,000 --> 00:29:10,000
I'm going to order me a boat
load, a lot of that primer. So, I'm

377
00:29:10,000 --> 00:29:13,000
adding, I better add a billion
molecules of that primer because I'm

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00:29:13,000 --> 00:29:16,000
going to make a billion copies
starting from such primers.

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00:29:16,000 --> 00:29:20,000
But if I have a billion copies of,
of number one and a billion copies

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00:29:20,000 --> 00:29:23,000
of number two and,
you know, these days,

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00:29:23,000 --> 00:29:26,000
billions aren't such big, you
know, molecules are Avogadro's

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00:29:26,000 --> 00:29:30,000
number and all that. It's
not hard to get things.

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00:29:30,000 --> 00:29:34,000
So, you throw in huge excess, a
massive excess of primer number

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00:29:34,000 --> 00:29:38,000
one, a massive excess of primer
number two, and you just do this.

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00:29:38,000 --> 00:29:42,000
Now, I mean, what does it cost
to make such a massive excess of a

386
00:29:42,000 --> 00:29:46,000
primer? It's about ten cents
a base, so it's two bucks,

387
00:29:46,000 --> 00:29:50,000
two bucks per primer give or take.
You know, so I can get you a better

388
00:29:50,000 --> 00:29:54,000
price if you want,
but, you know, anyway.

389
00:29:54,000 --> 00:29:58,000
It's not a bad price to, to
buy primer. So, you can just go

390
00:29:58,000 --> 00:30:01,000
out and order a pair of primers.
You can have them tomorrow.

391
00:30:01,000 --> 00:30:05,000
And then all you have to do
is add the primers to the,

392
00:30:05,000 --> 00:30:09,000
so I take DNA. Do I,
I, I need DNA from you.

393
00:30:09,000 --> 00:30:13,000
It turns out I could draw your
blood and purify DNA and all that.

394
00:30:13,000 --> 00:30:16,000
But it turns out that if all I
wanted to do was amplify one locus,

395
00:30:16,000 --> 00:30:20,000
I could actually take a Popsicle
stick and ask you to scrape the

396
00:30:20,000 --> 00:30:24,000
inside of your cheek. That'll
get enough cells off from

397
00:30:24,000 --> 00:30:28,000
the inside of your cheek, stick
it in a test-tube, and it'll

398
00:30:28,000 --> 00:30:31,000
actually have enough DNA there.
It turns out this is a very

399
00:30:31,000 --> 00:30:35,000
sensitive and powerful technique,
so, but before we get to that notice

400
00:30:35,000 --> 00:30:39,000
what we had to do. We
had to heat our DNA to 97

401
00:30:39,000 --> 00:30:43,000
degrees and add polymerase.
Then we heat again to 97, add

402
00:30:43,000 --> 00:30:46,000
polymerase, heat again to 97,
add polymerase. Why do I have to

403
00:30:46,000 --> 00:30:50,000
keep adding polymerase? Because
polymerase gets roomed at

404
00:30:50,000 --> 00:30:54,000
97 degrees so it's denatured.
So, the nuisance about PCR is I

405
00:30:54,000 --> 00:30:57,000
have to go to my Eppendorf
plastic tube, pop open the lid,

406
00:30:57,000 --> 00:31:01,000
stick in some DNA polymerase,
close it up, stick it back in a

407
00:31:01,000 --> 00:31:05,000
heating bath, let it go for a while,
take it out, pop it open, add some

408
00:31:05,000 --> 00:31:09,000
more polymerase, put it
back in the heating bath,

409
00:31:09,000 --> 00:31:13,000
pop it out. And this
is actually the way

410
00:31:13,000 --> 00:31:17,000
primitive scientists did PCR not
so long ago, OK? Wouldn't it be cool

411
00:31:17,000 --> 00:31:22,000
if we could engineer a DNA
polymerase that didn't denature at

412
00:31:22,000 --> 00:31:26,000
97 degrees? Because then what we
could do is just add the polymerase,

413
00:31:26,000 --> 00:31:31,000
close up the tube, put it
in a machine that goes heat,

414
00:31:31,000 --> 00:31:35,000
cool, heat, cool, heat, cool,
heat, cool, but you would have,

415
00:31:35,000 --> 00:31:40,000
so how do we, what kind of cleaver
biological engineering do we use to

416
00:31:40,000 --> 00:31:45,000
modify polymerase so it
won't denature at 97 degrees?

417
00:31:45,000 --> 00:31:49,000
Yes? Get it from a bacteria.
What kind of a bacteria would you

418
00:31:49,000 --> 00:31:53,000
ask for an enzyme that could work
in, in basically boiling water?

419
00:31:53,000 --> 00:31:57,000
Bacteria that basically live
in boiling water. Where would

420
00:31:57,000 --> 00:32:03,000
you look for such?
Thermal vents.

421
00:32:03,000 --> 00:32:09,000
You'll, geysers, things
like that. Life lives

422
00:32:09,000 --> 00:32:15,000
everywhere. What you go is
you find yourself a bacterium,

423
00:32:15,000 --> 00:32:21,000
so you find bacteria that lived in
geysers or in thermal vents and you

424
00:32:21,000 --> 00:32:27,000
purify their DNA polymerase. The
most famous one comes from the

425
00:32:27,000 --> 00:32:34,000
organism, the bacterium called
thermos aquaticus, aquaticus.

426
00:32:34,000 --> 00:32:38,000
Which of course means hot water,
right? That's what the bacteria is

427
00:32:38,000 --> 00:32:43,000
called, thermos aquaticus. And,
or, and its enzyme is called

428
00:32:43,000 --> 00:32:47,000
tack, tack. So, we'll
refer to it often,

429
00:32:47,000 --> 00:32:52,000
Taq polymerase, meaning from
this bacteria thermos aquaticus,

430
00:32:52,000 --> 00:32:57,000
OK? So, that's Taq. So, it turns
out that you can do this now without

431
00:32:57,000 --> 00:33:02,000
having to open and
close the test-tubes.

432
00:33:02,000 --> 00:33:11,000
Oops, I meant to put that here.
How sensitive is PCR? It's very

433
00:33:11,000 --> 00:33:20,000
sensitive. You could do,
so applications of PCR. Very

434
00:33:20,000 --> 00:33:30,000
versatile. First let's
just re-sequence a gene.

435
00:33:30,000 --> 00:33:36,000
Gene from yeast or from human.
You just need, you know, any DNA

436
00:33:36,000 --> 00:33:42,000
sample. Get my gene, get
my primers, and as I was

437
00:33:42,000 --> 00:33:48,000
indicating with a Popsicle stick,
I don't have to have it very pure,

438
00:33:48,000 --> 00:33:54,000
although in a laboratory you go
to the trouble of making it pure

439
00:33:54,000 --> 00:34:00,000
because you want it to
be pure and all that. Yes?

440
00:34:00,000 --> 00:34:06,000
Correct. Yeah. So,
remember I was making a fuss

441
00:34:06,000 --> 00:34:12,000
over the accuracy of replication,
right? And I said that on its own a

442
00:34:12,000 --> 00:34:19,000
polymerase might have an accuracy
to only about ten to the minus five.

443
00:34:19,000 --> 00:34:25,000
So, now, what were
the two mechanisms for,

444
00:34:25,000 --> 00:34:32,000
for repairing DNA,
for proofreading DNA?

445
00:34:32,000 --> 00:34:35,000
One was a built-in proofreading
activity that the enzyme had.

446
00:34:35,000 --> 00:34:38,000
The enzyme would have put in
a base, would check the base,

447
00:34:38,000 --> 00:34:42,000
and that actually helped by
an order of magnitude or two.

448
00:34:42,000 --> 00:34:45,000
And some of these polymerases
have a proofreading activity.

449
00:34:45,000 --> 00:34:49,000
But then we also discussed the
mismatch repair activity that would

450
00:34:49,000 --> 00:34:52,000
later come along and detect
mismatches. You're absolutely right,

451
00:34:52,000 --> 00:34:56,000
PCR is not as accurate as cells
because it doesn't have that

452
00:34:56,000 --> 00:35:00,000
mismatch repair activity. So,
when you take a PCR product,

453
00:35:00,000 --> 00:35:05,000
if I were to clone, so if I
were to take all the PCR product,

454
00:35:05,000 --> 00:35:10,000
say, from my beta-globin gene,
so I'm going to take my test-tube,

455
00:35:10,000 --> 00:35:15,000
I'm going to add my primers and
everything, I'm going to PCR,

456
00:35:15,000 --> 00:35:20,000
I'm going to PCR, and then I'm
going to get a lot of copies of

457
00:35:20,000 --> 00:35:25,000
beta-globin. If I were to take
that beta-globin and just directly

458
00:35:25,000 --> 00:35:30,000
sequence the DNA in the test-tube.
Here's my pieces of beta-globin.

459
00:35:30,000 --> 00:35:34,000
I can now sequence it by adding
a primer and doing my fluorescent

460
00:35:34,000 --> 00:35:39,000
sequencing and running it
on a sequencer and all that.

461
00:35:39,000 --> 00:35:44,000
Sorry, going the other way.
I'll run a sequencing reaction.

462
00:35:44,000 --> 00:35:48,000
I could actually do it, and what I
do it on is the whole population of

463
00:35:48,000 --> 00:35:53,000
a million or a billion molecules.
If any one of them is wrong it's

464
00:35:53,000 --> 00:35:58,000
going to be swamped out by
others, OK? Because I could do my

465
00:35:58,000 --> 00:36:03,000
sequencing reaction on
the whole PCR product.

466
00:36:03,000 --> 00:36:07,000
And random mistakes in one molecule
or the other will still be a tiny

467
00:36:07,000 --> 00:36:12,000
minority of the votes at any given
base, right? But suppose I were to

468
00:36:12,000 --> 00:36:17,000
take my PCR product, all
these amplified molecules here,

469
00:36:17,000 --> 00:36:22,000
and suppose I were to clone them
individually and I were to sequence

470
00:36:22,000 --> 00:36:27,000
each of those individual
clones instead of sequencing a,

471
00:36:27,000 --> 00:36:32,000
a mixture of all the
products. I would, in fact,

472
00:36:32,000 --> 00:36:36,000
see a higher mutation rate.
And you're absolutely right.

473
00:36:36,000 --> 00:36:41,000
When people clone PCR products they
have to check them afterwards and

474
00:36:41,000 --> 00:36:46,000
throw out the ones that are wrong,
OK? Absolutely right. Good, good,

475
00:36:46,000 --> 00:36:50,000
good. So, you guys are, you know,
right on top of the important issues

476
00:36:50,000 --> 00:36:55,000
about, about DNA. So, so I
can, I can take a gene and

477
00:36:55,000 --> 00:37:00,000
I can re-sequence it. I
can also do things like take

478
00:37:00,000 --> 00:37:05,000
blood and look for the
presence of a virus.

479
00:37:05,000 --> 00:37:11,000
So, I could re-sequence beta-globin
and study people and see who's got

480
00:37:11,000 --> 00:37:18,000
sickle cell anemia and all that.
I could take blood and I might want

481
00:37:18,000 --> 00:37:24,000
to say do I see the HIV virus
present in someone's blood?

482
00:37:24,000 --> 00:37:31,000
For example, HIV testing can be
done by making PCR primers for the

483
00:37:31,000 --> 00:37:37,000
sequence of the HIV
virus. It has a genome.

484
00:37:37,000 --> 00:37:41,000
Taking a human's blood sample and
PCR-ing it. If you get a positive

485
00:37:41,000 --> 00:37:45,000
PCR product, a PCR product that is
made by these two primers and if,

486
00:37:45,000 --> 00:37:49,000
for example, you checked that it,
that it gives you the HIV sequence

487
00:37:49,000 --> 00:37:53,000
then you know that that blood sample
has, that person has the HIV virus.

488
00:37:53,000 --> 00:37:57,000
This is a way to do this. The
PCR reaction itself is fast.

489
00:37:57,000 --> 00:38:01,000
Typically takes hours. In
fact, can be forced to go much

490
00:38:01,000 --> 00:38:04,000
more quickly by machines
that rapidly thermocycle.

491
00:38:04,000 --> 00:38:08,000
And you can actually PCR in five
minutes, although people don't do it

492
00:38:08,000 --> 00:38:11,000
very often, but if you put a
thin glass capillary and go heat,

493
00:38:11,000 --> 00:38:14,000
cold, heat, cold very, very
quickly, there's a machine from Idaho

494
00:38:14,000 --> 00:38:18,000
Technologies that can do it in five
minutes, but it's usually not the

495
00:38:18,000 --> 00:38:21,000
trouble. And you just put it in and,
you know, in a couple hours you'll

496
00:38:21,000 --> 00:38:24,000
get an answer there as to
whether or not somebody has HIV,

497
00:38:24,000 --> 00:38:28,000
for example. So, you can do
that to detect relatively low

498
00:38:28,000 --> 00:38:33,000
quantities of virus.
How low can you go?

499
00:38:33,000 --> 00:38:39,000
Well, it turns out, what's
the limit? What's the

500
00:38:39,000 --> 00:38:45,000
smallest number of molecules you
might be able to detect in a sample?

501
00:38:45,000 --> 00:38:51,000
Theoretically. One. You
can't fewer than one molecule,

502
00:38:51,000 --> 00:38:57,000
right? So, one might be the limit.
So, how could I arrange to have a

503
00:38:57,000 --> 00:39:01,000
single molecule in a test-tube?
I would like to have a test-tube

504
00:39:01,000 --> 00:39:04,000
that has exactly one copy
of the beta-globin gene.

505
00:39:04,000 --> 00:39:08,000
What, how's the best, what's
the best way to get exactly

506
00:39:08,000 --> 00:39:11,000
one copy of beta-globin
and put it in the test-tube?

507
00:39:11,000 --> 00:39:14,000
Sorry? You can't.
Why? Just one molecule.

508
00:39:14,000 --> 00:39:18,000
I want to get exactly one
copy of beta-globin. I could,

509
00:39:18,000 --> 00:39:21,000
I could just take total DNA
and dilute it so, on average,

510
00:39:21,000 --> 00:39:24,000
there's only one copy. Or,
actually, is there any way to,

511
00:39:24,000 --> 00:39:28,000
I mean can I, I'd just like to
buy a package that contains exactly

512
00:39:28,000 --> 00:39:32,000
one beta-globin. Sorry?
Bind it to something big.

513
00:39:32,000 --> 00:39:36,000
Let's think biologically. Does
biology package up a single copy of

514
00:39:36,000 --> 00:39:41,000
beta-globin? Sorry?
Gametes. How about a sperm?

515
00:39:41,000 --> 00:39:46,000
Let's grab a sperm by its tail
here, put it in the test-tube.

516
00:39:46,000 --> 00:39:50,000
It's one copy of beta-globin.
So, you can actually take cell

517
00:39:50,000 --> 00:39:55,000
sorters and have it cell sort
sperm into individual test-tubes.

518
00:39:55,000 --> 00:40:00,000
You now know there's
one copy of beta-globin.

519
00:40:00,000 --> 00:40:05,000
Heat it up, it will crack open
the sperm, add your primers,

520
00:40:05,000 --> 00:40:10,000
you can amplify beta-globin, it's
a single copy. That proves its

521
00:40:10,000 --> 00:40:16,000
extraordinary sensitivity. You
can do it with a single sperm.

522
00:40:16,000 --> 00:40:21,000
You can do it with a single
egg also, but harder to come by.

523
00:40:21,000 --> 00:40:27,000
So, with that level of sensitivity,
you could do the following. So,

524
00:40:27,000 --> 00:40:32,000
single sperm typing. Now,
single sperm typing is cool but

525
00:40:32,000 --> 00:40:36,000
sort of useless. What are
you going to do with it,

526
00:40:36,000 --> 00:40:41,000
right? But here's another thing
you could do. Embryo typing.

527
00:40:41,000 --> 00:40:45,000
Suppose someone has a genetic
disease in their family,

528
00:40:45,000 --> 00:40:50,000
maybe it's Huntington's disease.
And suppose that the individual with

529
00:40:50,000 --> 00:40:54,000
Huntington's disease wants to
have kids. Or the individual,

530
00:40:54,000 --> 00:40:59,000
sorry, the individual who is at risk
for Huntington's disease or breast

531
00:40:59,000 --> 00:41:04,000
cancer or whatever
wants to have kids.

532
00:41:04,000 --> 00:41:13,000
What you can do is with an in vitro
fertilization clinic you're able to

533
00:41:13,000 --> 00:41:23,000
obtain eggs, fertilize eggs in vitro,
and grow them up in a Petri plate to

534
00:41:23,000 --> 00:41:33,000
8 or 16 cell stage before
re-implanting embryos

535
00:41:33,000 --> 00:41:41,000
back in the mother. Wouldn't
it be cool if we could

536
00:41:41,000 --> 00:41:48,000
choose to only re-implant an
embryo that did not have the genetic

537
00:41:48,000 --> 00:41:56,000
disease? How are we going
to do that? PCR. How are we,

538
00:41:56,000 --> 00:42:02,000
so what do we do?
We take the embryo.

539
00:42:02,000 --> 00:42:06,000
We make DNA from the embryo. We
do PCR and we say, ah-ha, this

540
00:42:06,000 --> 00:42:10,000
embryo did not have the genetic
disease. Problem is it has killed

541
00:42:10,000 --> 00:42:15,000
the, the cells there,
right, it killed the embryo.

542
00:42:15,000 --> 00:42:19,000
Any ideas? Pull off one cell.
Remove a single cell. It turns out

543
00:42:19,000 --> 00:42:24,000
that at stage the cells
are not differentiated.

544
00:42:24,000 --> 00:42:28,000
If I remove one cell from an
embryo at that very early stage,

545
00:42:28,000 --> 00:42:33,000
the other cells with make a
perfectly happy, healthy baby.

546
00:42:33,000 --> 00:42:37,000
That cell is not necessary.
This single cell sensitivity is

547
00:42:37,000 --> 00:42:41,000
very valuable because I can actually
do single cell genotyping on in

548
00:42:41,000 --> 00:42:45,000
vitro fertilized embryos and
be able offer parents a chance,

549
00:42:45,000 --> 00:42:49,000
the opportunity to re-implant only
those embryos that do not have the

550
00:42:49,000 --> 00:42:53,000
genetic defect. That's
cool. That's really cool.

551
00:42:53,000 --> 00:42:57,000
There are other things you might
be able to do. If you're treating a

552
00:42:57,000 --> 00:43:01,000
patient with cancer, a
patient, a cancer patient and

553
00:43:01,000 --> 00:43:05,000
you've given chemotherapy you want
to know have I managed to eradicate

554
00:43:05,000 --> 00:43:11,000
the cancer cells? And six
months later have any of the

555
00:43:11,000 --> 00:43:19,000
cancer cells come back?
I could look for very low

556
00:43:19,000 --> 00:43:27,000
quantities of cancer cells. I
can, I can do surveillance for

557
00:43:27,000 --> 00:43:35,000
low quantities of cancer
cells following chemotherapy.

558
00:43:35,000 --> 00:43:39,000
And, of course, I
can also do forensics.

559
00:43:39,000 --> 00:43:44,000
I could take a small sample of
blood from the scene of a crime or

560
00:43:44,000 --> 00:43:49,000
saliva from the back of an
envelope that someone has licked,

561
00:43:49,000 --> 00:43:53,000
and I could do PCR and look for
genetic variations that distinguish

562
00:43:53,000 --> 00:43:58,000
people. And, presumably, you
see all that stuff on television

563
00:43:58,000 --> 00:44:04,000
all the time. So, that's
what PCR is good for.

564
00:44:04,000 --> 00:44:10,000
It's good. All right. Last topic,
very brief topic, but I do want to

565
00:44:10,000 --> 00:44:17,000
mention. This was being able to
analyze a gene directed mutagenesis.

566
00:44:17,000 --> 00:44:23,000
And I won't go through the details
of all this, but I just want to at

567
00:44:23,000 --> 00:44:30,000
least basically
describe the concept.

568
00:44:30,000 --> 00:44:36,000
I could take any piece of DNA,
say from a drosophila, and I can

569
00:44:36,000 --> 00:44:42,000
mutate the DNA in vitro. I can
change this base from a G to

570
00:44:42,000 --> 00:44:48,000
a C. There's a right,
there's a proper protocol and

571
00:44:48,000 --> 00:44:54,000
cooking trick for doing that. It
involves putting a certain oligo

572
00:44:54,000 --> 00:45:00,000
over it and extending, and
it doesn't matter exactly how.

573
00:45:00,000 --> 00:45:04,000
I could insert an extra gene
into that. I could use a little

574
00:45:04,000 --> 00:45:08,000
restriction enzyme to open
it up and stuff something in.

575
00:45:08,000 --> 00:45:13,000
I could delete something from this.
Maybe I'll use a restriction enzyme

576
00:45:13,000 --> 00:45:17,000
to cut it open, et
cetera. Basically I could,

577
00:45:17,000 --> 00:45:22,000
I can fuse genes together. I can
do whatever kind of construction of

578
00:45:22,000 --> 00:45:26,000
pieces of DNA and modifications
of pieces of DNA that I would

579
00:45:26,000 --> 00:45:31,000
like to do in vitro. I can
then take that mutated gene,

580
00:45:31,000 --> 00:45:36,000
let's say the gene is an
enzyme, encodes an enzyme,

581
00:45:36,000 --> 00:45:41,000
and the enzyme has an active site.
I could change the code for the

582
00:45:41,000 --> 00:45:46,000
amino acid right at the active site
to see if that amino acid really

583
00:45:46,000 --> 00:45:52,000
matters or not. I can
do any of those things.

584
00:45:52,000 --> 00:45:57,000
And I can put this back in an
organism. Remember that you,

585
00:45:57,000 --> 00:46:02,000
I said you could transform
DNA back into bacteria?

586
00:46:02,000 --> 00:46:07,000
Well, you can also do such
things as simply inject DNA into

587
00:46:07,000 --> 00:46:11,000
a fertilized egg. In fact,
at the stage where there's

588
00:46:11,000 --> 00:46:15,000
a male and a female pronucleus
that haven't fused yet right after

589
00:46:15,000 --> 00:46:19,000
fertilization. You can
take your little pipette

590
00:46:19,000 --> 00:46:22,000
and a needle and you can inject some
of the DNA you want into the male

591
00:46:22,000 --> 00:46:26,000
pronucleus, and then when the male
pronucleus and the female pronucleus

592
00:46:26,000 --> 00:46:30,000
fuse and the embryo grows
it will have your DNA.

593
00:46:30,000 --> 00:46:36,000
You can make mice that carry
whatever gene you've modified like

594
00:46:36,000 --> 00:46:42,000
this. You can also not, you,
you can also not just modify a

595
00:46:42,000 --> 00:46:49,000
piece of DNA and add,
this is gene addition,

596
00:46:49,000 --> 00:46:55,000
you can also do gene subtraction.
You can do gene subtraction and,

597
00:46:55,000 --> 00:47:01,000
again, I won't worry about
the details here, by taking

598
00:47:01,000 --> 00:47:07,000
embryonic stem cells. Much
in the news these days,

599
00:47:07,000 --> 00:47:11,000
and we may come back to them.
And in vitro, working with

600
00:47:11,000 --> 00:47:15,000
embryonic stem cells, to
transform a piece of DNA that has

601
00:47:15,000 --> 00:47:19,000
been arranged to recombine into the
gene of interest and know it out.

602
00:47:19,000 --> 00:47:24,000
So, if you build, if you build a
piece of DNA in vitro and you put it

603
00:47:24,000 --> 00:47:28,000
into a whole bunch of embryonic
stem cells you can select,

604
00:47:28,000 --> 00:47:32,000
by various cleaver techniques,
for those embryonic stem cells that

605
00:47:32,000 --> 00:47:38,000
have taken up your gene. And
not just taken it up but slammed

606
00:47:38,000 --> 00:47:45,000
it into the normal locus in
place of the normal locus.

607
00:47:45,000 --> 00:47:52,000
And that way you can knock out
a gene. You can do gene knockout.

608
00:47:52,000 --> 00:47:59,000
So, the basic point of this now,
to summarize these many lectures is

609
00:47:59,000 --> 00:48:06,000
we're now at the point where
this picture that we saw at the,

610
00:48:06,000 --> 00:48:13,000
at the beginning, function, gene,
protein, that we understood now

611
00:48:13,000 --> 00:48:20,000
first as a methodology,
genetics, biochemistry.

612
00:48:20,000 --> 00:48:26,000
And then we understood how genes
encode proteins through molecular

613
00:48:26,000 --> 00:48:32,000
biology. These tools of
recombinant DNA allow us to move

614
00:48:32,000 --> 00:48:37,000
in any direction. You want
to find the gene underlying

615
00:48:37,000 --> 00:48:41,000
a function, find the gene
for Huntington's disease?

616
00:48:41,000 --> 00:48:45,000
We could do it. Clone it
based solely on its linkage.

617
00:48:45,000 --> 00:48:49,000
You want to find the gene encoding
a protein? If I know its amino acid

618
00:48:49,000 --> 00:48:53,000
sequence, I can find the DNA
sequence that corresponds it.

619
00:48:53,000 --> 00:48:57,000
If I want to find what a certain
protein does, its function,

620
00:48:57,000 --> 00:49:01,000
I could get the gene for that
protein. I could knock out the gene

621
00:49:01,000 --> 00:49:05,000
for that protein and
see what its function is.

622
00:49:05,000 --> 00:49:08,000
Suddenly, for the
mathematicians amongst the group,

623
00:49:08,000 --> 00:49:12,000
this becomes a commutative diagram,
which you can chase around in any

624
00:49:12,000 --> 00:49:15,000
direction. That is, in a
sense, what the 20th century

625
00:49:15,000 --> 00:49:19,000
was about, was intellectually these
two disciplines merging through

626
00:49:19,000 --> 00:49:22,000
molecular biology and then
recombinant DNA giving you all the

627
00:49:22,000 --> 00:49:26,000
tools that if you're sitting at any
place in this triangle you can move

628
00:49:26,000 --> 00:49:29,000
this way and that way,
from a gene to a protein,

629
00:49:29,000 --> 00:49:33,000
from a protein to a gene, from
a function to a gene, from a

630
00:49:33,000 --> 00:49:36,000
function to a protein. Much
of the rest of the course we'll

631
00:49:36,000 --> 00:49:40,000
talk about how you use these tools,
but this brings to a close this

632
00:49:40,000 --> 00:49:44,000
first chunk of the course about the
concepts and the methodologies of

633
00:49:44,000 --> 00:49:48,000
molecular biology. Now, if
you hang on one more minute,

634
00:49:48,000 --> 00:49:52,000
this is my last lecture for a while.
I won't be, we're having an exam on,

635
00:49:52,000 --> 00:49:56,000
we have a quiz on Monday, and
then Bob's taking over again.

636
00:49:56,000 --> 00:50:00,000
So, I won't see you for the
next week or so. So, two things.

637
00:50:00,000 --> 00:50:04,000
One, I won't see you before the
World Series is over so everyone

638
00:50:04,000 --> 00:50:09,000
please think good thoughts
about the Red Sox. Number two,

639
00:50:09,000 --> 00:50:13,000
I will not see you
before the election. Vote.

640
00:50:13,000 --> 00:50:18,000
It's your choice who you
vote for, but vote. Good-bye.