[ Silence ]
>> OK, so, hopefully
that wasn't too painful.
I realize it might have been--
if you even put your name
on the piece of paper,
you will get some points
that I promise you.
In addition, next time I say
that there's something I'd
like you go look up, I hope
you take I mores [phonetic]--
you take it seriously.
But don't panic.
Hopefully, that's
not too painful.
Let me just tell you very
quickly the answers, OK?
So, last time we saw
that half life of DNA was
on the order of 220
million years.
And I challenge you
to go out and find
out what it-- had to measure it.
And at least one person came to
my office hour with the solution
which is to heat the DNA
so you can heat it up
and to the high temperature,
measure the half life
at the higher temperature
and extrapolate back
to the lower temperature.
And you don't have to provide
me with a lot of equations
to show that that's true.
But if you just write
heat on probably--
number two, I will
accept it, OK?
But again, if you have your
name on a sheet of paper
with your UCI ID
number, we'll give--
you'd get at least
some points, OK?
OK. All right, we're
back to normal stuff now.
I want to pick up where
we left off last time.
We were talking about structure
of DNA and reactivity of DNA,
and we saw last time-- oh gees.
What happened here?
One moment.
I just [inaudible] this.
OK, we saw last time a lot
about Watson-Crick based-pairing
and structures of DNA.
And it's B-DNA form.
Now, one thing I got
asked about immediately
after the lecture was, you know,
ethidium bromide is commonly
considered to be a carcinogen.
This molecule over here that
we discussed as an interculator
of DNA is also a carcinogen.
It's a molecule that causes
cancer and that's the reason why
when we work with it in
the laboratory, over here,
we're exceptionally careful to
keep it off of our skin, OK?
Now, yeah, it's a-- it
articulates into DNA
and DNA interculators
can caused,
can help to initiate
carcinogesis, can--
it helps initiate cancer
in the following ways.
Number one, it distorts
the structure of DNA.
I showed you that on the
previous slide over here
where I showed you how the
structure of the DNA has
to unwind to accept
interculator.
So it distorts the
structure of DNA.
Number two, it places a
hydrophobic functionality
in the center of the DNA.
This hydrophobic functionality
can inappropriately attract
transcription factors to the
DNA setting off incorrect
transcription, OK?
So those are two possible modes
that this can start to cause--
this can start to initiate
improper self responses
that will eventually
lead to cancer.
And I want to talk
about cancer today.
It's one of the prime
topics in this class.
It's something we've
already spend quite a bit
of time discussing in
many different context
and we'll certainly
be talking about it
in the context of DNA, OK?
So, any other questions
about what we saw last time.
That was a really good question.
OK, last thought about
ethidium bromide,
although it is definitely--
it is a cancer causing agent
and it's something you
definitely do not want to ingest
or you definitely want to
keep it off your hands.
I don't want to exaggerate
its carcinogenic potential.
It's also used as an
antibiotic in sheep.
I don't know if it's still
is, but it was for a while.
It is-- It does have
some other--
and so it's actually
fed to sheep.
I don't know why sheep.
But in any case, as a
tumor forming agent,
its activity is rather
modest specially compared
to the other cancer causing
small molecules that we're going
to see very shortly, OK?
All right, well let's
dive right in this.
So last time, I was
showing you that DNA likes
to form Watson-Crick
based-pairing.
This is C binding to Gs.
A is binding to Ts
and we saw that GCs,
Watson-Crick based-pairings
have three hydrogen bonds
and ATs has only have two, OK?
Now, if we know that AT
based-pairs are weaker than GCs,
there's a very simple that we
can use to estimate the strength
of any two DNA strands
taking together.
This rule is called the
Wallace Rule and I'd
like you to memorize it, OK.
The Wallace Rule tells us
that the approximate
melting temperature
for DNA sequence is equal
to 2 times the number
of AT base pairs plus 4 times
the number of GC base pairs
in degree C. This melting
temperature is where 50 percent
of the DNA is no longer forming
a double-stranded structure.
Let me explain.
So0 when you do a UV/Vis
scan of DNA, this is what--
here's what you find, OK?
So this is absorbance on
the Y axis and wave length,
the nanometers on the X axis.
And this is single-stranded DNA
and then this is
double-stranded DNA in blue.
And to get this, you simply
add higher temperature.
So at higher temperature,
the DNA stands melt apart.
In other words, they separate
out into the two single strands
and so at 82 degrees, this
is the single-stranded
and 25 degrees, this is
the double-stranded DNA.
Notice that the absorbance
at 260 nanometers is higher
for the single-stranded DNA
than the double-stranded DNA.
So you can use that change in
absorbance to follow whether
or not your DNA is
single-stranded
versus double-stranded.
And so, you can do this
while at the same time,
you ramp up the temperature
and at
about 50 percent--
and so here it is.
Pure double-stranded DNA, lower
absorbance and then here it is
at a higher temperature
where it's entirely
single-stranded DNA.
And the approximate
50 percent part
where 50 percent is melted is
called the melting temperature.
And again, you can estimate
what this melting temperature is
using this Wallace rule formula.
People in chemical biology
laboratories uses Wallace rule
formula on a daily basis, OK,
certainly in my laboratory
and certainly and probably five
or six other laboratories
here at UCI Irvine.
So, I'd like you to
memorize this rule.
It's incredibly useful.
Now, you're probably
wondering, big deal.
So I know whether or not
something melts, whether
or not it forms double
stranded DNA.
If you know the temperatures
that it forms double-stranded
DNA, you can start
to design structures
made out of DNA.
Let me show you.
OK. So here are structures of
DNA where it's single-strands
of DNA that are now
hybridizing against each other
and forming elaborate patterns
such as this pattern here.
And here's an atomic
force microscope image
of this double-stranded DNA
and you could see it's all--
it's forming this
exact costly pattern
that was designed using
something just a little bit more
complicated than
the Wallace rule
which I asked you
to memorize, OK?
It gets even better.
Check this out.
OK. So this is worked done by
Paul Rothemund and colleagues
and coworkers at Caltech.
And he's using the Wallace
rule to design DNA that folds
up into happy faces over
here or check out this map
of the world written out
of DNA that's been folded
up with itself.
OK. So that Wallace
rule that I asked you
to memorize is actually
pretty powerful.
You can develop whole
structures of that stuff.
Now, exactly what the structures
of DNA are going to be useful
for is not a hundred
percent known at this point.
There are sort of a frontier
in chemical biology building
structures out of DNA
and then trying to do
something useful with them.
I've only seen one paper
in 20 years of staring
at these beautiful pictures
that has convinced me
that maybe there might be
something useful about this.
And in that paper this DNA--
a DNA structure like this one,
not the happy face but
something elaborate was used
as a delivery vehicle that
bound to the surface of cells
and then dislodged
drug therapeutic.
And it's possible that our
future might feature many more
of these sort of examples of
nanometer scale structures
that are designed by you,
by the people on this room
to have specific
properties such as binding
to specific cell
types, unloading cargos
at specific times, et cetera.
This is a really exciting
frontier and I encourage you
to think about it in
your proposal preparation
because it's an area
that's kind of wide open
for creative-- for creativity.
OK, so in addition to those
sort of macro structures of DNA
that we saw, short
stretches of DNA can also fold
and there's a couple
of canonical structures
that we're going to
see time and again.
One of these for example is
called-- are called hairpins.
One of these is called
the hairpin.
And so, these consist
of a sequence
that folds back on itself.
Notice that it satisfies all
of the Watson-Crick base pairing
requirements, G is to Cs.
A is to Ts and it forms
something that looks kind
of like an old-fashioned
hairpin.
OK. And it looks
structurally like this.
This is the x-ray crystal
structure of what it looks like.
And again, we're going
to see this quite a bit.
OK. So DNA has a
propensity to fold on itself.
It wants to form
Watson-Crick base pairs.
It wants to form Watson-Crick
base pairs with other sequences.
It wants to form base
pairs with itself.
And so for this reason,
DNA is rarely found.
It's sort of an unwound
which rarely found a
single-stranded DNA
for one thing.
And furthermore, it's
rarely found in the sort
of canonical B-DNA conformation
that I've been showing you
where it says nice right-handed
coiled double helix.
Rather, in cells, we
typically find DNA in a wound
up configuration
called a supercoil.
So a supercoil is
where you take a coil
such as this old-fashioned
telephone cord
which I'm sure is unfamiliar
to everyone in this room.
But back in my day kids,
we used to have this
and it would form the
sort of supercoils
and it drove you nuts.
You know, you constantly
be on the phone trying
to untangle the darn thing.
In fact, she [phonetic] was
kind of a nice thing to do
because if you're on the phone
with the tense conversation
or something, it gave you a
task to take your mind off
of the annoyances that you're
dealing with on the phone.
But anyway, so this
is called supercoiling
and this is an example of a
DNA plasmid which is a sequence
of DNA that forms a circle.
OK. So this is a nice plasmid
DNA and again we rarely find it
in this sort of B-DNA where
it's completely unwounded
configuration, rather
it likes to twist up.
So here's a little twistiness.
Here's more twistiness,
even more twistiness
and then finally even--
the most twistiness.
OK. So that's really the
structures of DNA that we find.
For short sequences,
we find it wound
up with itself to form hairpins.
I showed you that
structure first.
For larger sequences
like plasmids,
we find it supercoiling
and twisting itself up.
And then for even larger
sequences like you carry
out at genomes which I'll
show you on the next slide,
it gets even more
twisty than that.
OK. So-- oh, before
I get to that,
here's why it has
to get so twisty.
This is one of the benefits of
having it coiling up on itself.
This is just the DNA in
a single E. coli cell.
This is a classic
picture of that DNA
where really toward [phonetic]
the force microscopy was used
to lice the cell and
spew out all of its DNA
and you could see it's just
an enormous amount of DNA
for such a small cell.
And the human cells face a
similar compaction problem,
right.
The human genome
would be a rough--
roughly 1.7 meters if
completely unwound.
So this property to
want to wind on itself,
to coil up with itself is
actually a really important one.
And so, when we look
at human DNA,
we find that it's compacted
up into chromosomes.
I'm sure-- hopefully, this
is not an unfamiliar concept.
In fact actually, I showed
you examples of chromosomes
from I believe it was
guinea pigs when we talked
about bromouracil
earlier this week.
In any case, so here're
some images
of human chromosomes over here.
And here's how it's
compacted up.
So, the DNA, here's the B-DNA
that we've been looking
at over here.
I admit this is a terrible
rendering because it looks
like it's a single helix.
Note that it's lacking
the major groove.
This is-- or the
lacking the minor groove.
This is one of my pet peeves
about artistic depictions
of DNA.
But I'm going to let it
slide by at this moment.
In any case, so here's
the regular B-DNA.
The B-DNA is going to wrap
around the protein
structure called the histone
and this is going to act
like the spools for thread.
And then these histones
are going to coil up
and those coils are
going to coil up further
until eventually you get
to something that's massively
compacted into a chromosome.
Now, the problem of course
and this is the problem
if you're on the phone as well.
So imagine you're on the phone
with these old-fashioned
telephone cords,
what you find is it has very
hard to untangle the DNA
without disconnecting the cord
and making a little break in it.
OK. And that's one of
the annoying things
about supercoiling of
old-fashioned telephone cords.
Similarly with DNA, where you
have this one 1.7 meter long
object and a 20 microne long
cell, there has to be a solution
to uncoil the DNA
and the solution is
to make transient breaks.
So shortly breaks in the
DNA using an enzyme called
DNA gyrase.
And here's an example of this.
This is a DNA gyrase that acts
kind of like scissors, OK?
So, notice that it's a dimer.
The two arms down here can open
up and the thing could just grab
onto the DNA and then
introduce two breaks
on the DNA allow the super--
the DNA to relax, to uncoil
and then it gets rejoined.
This turns out to
be a Achilles' heel
for bacterial cells,
for cells in general.
In other words, it's a
spot that can be targeted
with the antibiotics and
we'll be talking about this.
We'll be talking quite a
bit about different ways
that antibiotics works.
So antibiotics are
pharmaceuticals, therapeutics
that are given to patients
to eliminate bacterial
infections or fungal infections.
In this case, this is a really
effective antibiotic that's
giving quite a bit.
This is the antibiotic Cipro
which I'm sure many of you
in this classroom have
taken at one point.
I read somewhere that like 85
percent of American women come
down with a UTI, a urinary
tract infection at some point,
the first line of
antibiotic used
against that is often Cipro.
And Cipro works by inhibiting
the DNA gyrase of bacteria.
OK. Here's another one as well,
another inhibitor as well.
OK, so let's talk
a little bit more
about these bacterial plasmids
because I want to transition
into a discussion of
biotechnology and cutting
and pasting DNA in large scale.
So, oftentimes,
DNA is transferred amongst
organisms using plasmids.
Plasmids are short
circular stretches of DNA.
They need to have-- all plasmids
need to have two properties, OK?
They must have sequences
that encode two items.
Item number one is an
origin of replication.
The origin of replication
abbreviated ORI is the spot
that somehow convinces the cell
that's taking up the plasmid
to start transcribe-- or start
replicating that DNA, OK.
So that origin of replication
kicks off replication
of the plasmid.
Without that, the plasmid
would just be there
and it wouldn't get copied,
and it wouldn't get passed
on to the next little guy, OK?
So that's absolutely essential.
The other essential thing
is that the plasmid has
to confer some advantage
onto the new host, OK?
In other words, the new
bacteria has to take it
up and say, "Oh, yeah.
This is useful."
Otherwise, the plasmid will
get quickly shunted aside
because cells are under
a lot of pressure.
They have a lot of works to do
and they have a limited amount
of resources, carbon, nitrogen,
oxygen, things like that
that are available to do all of
the priorities that they have.
OK, and that's kind of a long
winded way to say that this has
to confer some resistance
oftentimes
to some sort of antibiotic, OK?
So, resistance markers
are sequences of DNA
that encode a protein
that confers resistance.
OK. So for example, you can
have a resistance marker
that encodes resistance to
the antibiotic tetracycline
and this gene will work
by actively pumping tetracycline
out of the cell, OK?
So, when the-- when the
cell takes up this plasmid,
it's going to synthesize this
pump that goes to the surface
of the cell and then every time
it gets tetracycline it just
pumps it out of the
cell furiously
and that allows the
cell to live.
So only the cells that have
the plasmid will survive an
onslaught of the
antibiotic tetracycline
which again is a very
common antibiotic
that I mentioned a few
of you have encountered.
It's often used, for example,
I believe for acne treatment.
OK. Here are some other classic
examples of other antibiotics
that are used in my laboratory
and other chemical
biology laboratories
as selection markers
for drug resistance.
And the way this works
is will coat the cells,
the bacterial cells on a plate
and the plate has an agar
which I'll show you the
structure very shortly.
It's isolated from seaweed.
It's a basically just a polymer
and inside this agar plate,
we'll have some concentration
of one to these antibiotics.
And so the only colonies
and each one
of these circles is
a colony that appears
on this plate are colony or
bacteria cells that have taken
up the plasmid because now,
those cells are resistant
to the antibiotic.
OK, so here is another
way that this can work.
So, antibiotic that's commonly
used is chloramphenicol.
Chloramphenicol inhibits
the ribosome.
We talked about the
ribosome before.
The drug resistance gene encodes
an enzyme called chloramphenicol
acetyltransferase or CAT
and this enzyme transfers
an acetyl group
to a primary hydroxyl
of chloramphenicol, OK?
So, here's the acetyl group
and acetyl-CoA and it's going
to get transferred to this
primary hydroxyl in a reaction
that essentially disarms the
chloramphenicol preventing it
from binding to the ribosome and
allowing the cells on this plate
of chloramphenicol to live.
Third, a very common
resistant marker, OK,
so I've shown you tetracycline.
We've talked about
chloramphenicol.
Third one, the third one
are beta-lactam antibiotics
of these sort.
Notice that this
is a beta-lactam.
A lactam, of course, is a cyclic
ring that has an amid bond
on it, and this is beta because
it has two carbons, alpha, beta.
And so, that's the beta-lac--
that's the origin of the
beta lactam nomenclature
which I know we talked
about in 51C.
Hopefully, you encountered
as well.
In any case, beta-lactamase
is a--
enzyme encoded by the
beta-lactamase gene
that confers the ability
to hydrolyze this
amid bond that's part
of the beta-lactam ring.
And this is a very common gene
that's found out on environment.
So, you can probably
scoop up, you know,
some dirt over here just
outside Rowland Hall
and you can readily find
this beta-lactamase gene.
And so for this reason,
medicinal chemists are
constantly making new
antibiotics that avoid that
environmental drug resistant
that sort of omnipresent, OK.
So for example, here
are two different kinds
of beta-lactam based antibiotics
and notice the structural
differences.
This one has this benzoyl
functionality over here.
This one has a carboxylate--
carboxylic acid and a
phenol group instead.
And so, all of those
little differences change,
affect the ability of the
drug resistance enzyme,
the enzyme conferring
drug resistance to bind
to the antibiotic and
hydrolyzes them and bond.
Maybe this carboxylate
sticks into the protein
and prevents the binding
and that's a useful thing.
OK. So we're constantly on
the hunt for new antibiotics
because the antibiotics we have
seem to allow very rapid risk--
evolution of drug
resistance and so,
there's a constant need
really for our society
to develop new classes
of antibiotics
that are more effective than
the previous generation.
And in the last 10 years or so,
there's been a real renaissance
of research in this area
to develop even more
effective antibiotics.
OK. Let's get back to our
discussion of DNA structure.
I showed you structure
of plasmids.
Here's structure of
a eukaryotic genes,
eukaryotic DNA that's wrapped
around nucleosome, et cetera.
I don't have very much
more to say about that.
Let's take a closer look however
at the structure
of these histones.
So, the histones are these
hexameric proteins shown here
in yellow and green
where in green,
these are positively
charged residues, OK?
So those are residues whose
positive charge can interact
with negatively charged
phosphodiester backbone
of the DNA.
Charge-charge interaction.
Nice long range interaction.
This wrapping up though
basically hides the DNA
and prevents it from
being transcribed.
When it's wrapped up around
the histone it can't be a read
out and, you know,
use for transcription.
And so, basically, whether or
not the histone is wrapping
up things, it's-- it
controls transcription
and controls packaging.
So these proteins over here
are very tightly regulated
as to whether or not they're
going to be binding to the DNA
and one easy way to
do this regulation is
to acetylate the lysines side
chains, OK, and I'll show you
that on the next slide.
OK. So first, this is
the structure of lysine.
Lysine has a primary mean
and here's lysine
within acetyl group.
What do you think the charge is
if something has a primary mean
at neutral pH which
is the pH roughly
of the cell approximately?
So it has a primary
mean functionality
in neutral pH. What
is its charge?
[ Pause ]
I'm a very patient guy.
[ Laughter ]
>> Positive one.
>> Positive.
>> Positive.
Positives.
Very good.
OK, good. So, when-- So
if this is bear lysine,
it will have a positive charge
and acetyl group over here,
it is back to neutral, OK?
So this guy, positive
charge, acetyl group, neutral.
So that controls whether or
not that lysine, the amino acid
of the protein interacts
with the DNA.
If it's positive charge,
it's like a homing
beacon for DNA, right?
The DNA is negatively charged.
Two of these want
to stick together.
If it's acetylated
however, it's not going--
it's going to be neutral and
the two are not going to want
to interact with each other.
Here's one that's even wilder.
In this case, you're
taking the primary amine
of lysine side chain and turning
it into a secondary amine
or tertiary amine or
even a quaternary amine.
And when you do this,
you're making the
lysine side chain fixed
as a positive charged.
OK, now I should say, it's
not fixed permanently.
It used to be thought
that it is but now we know
that actually this is a
reversible modification
as this is acetylation, OK?
So, this case, it's binding to
DNA, binding to DNA and then
when you get rid of these
methyl groups, it's back to--
it's still bonded to DNA but
then it can get acetylated
so it's no longer
binding to DNA.
OK. So there is a whole series
of different modifications
to the surface side chains,
the surfaces of the histones.
And all of these modifications
have an important consequences,
OK.
So for example, some
of these modifications
like these larger ones down
here direct the histones
into the proteosome which is
basically the garbage disposal
for the cell and so
those get flushed away
and thrown into the trash.
And then others like
this phosphorylation
of a hydroxyl functionality
found on the surface
of the DNA can regulate the
structure of the histone as well
and perhaps interfere
with this binding
to the negatively charged DNA.
OK. So, all of this stuff
is tightly choreographed,
there are enzymes
that add each one
of these modifications
highlighted
in blue on the slide.
And those enzymes are going to
control its binding affinity
for DNA and in turn control
whether or not the DNA is hidden
or available for transcription.
And you can imagine, this is
very tightly choreographed
by the cell.
If anything gets in
there to mess stuff up,
all kinds of havoc
can be wreck, right?
Because the cell has to control,
you know, turning on, you know,
specific genes has
specific times, right.
You would not want, for
example, you know, a muscle cell
to suddenly start growing, I
don't know, neurons or some--
you know, the genes that are
required for neuron growth,
neurite growth or
something like that.
That would be really bad, OK?
So everything is very tightly
choreographed at this level.
All right.
There are of course
small molecules
that inhibit these
histone deacetylases.
I shouldn't say of course.
This is actually a
discovery that was made
by Jack Toden [assumed spelling]
who is a graduate student--
when I was a graduate student,
the same lab where I was.
This discovery is made when
I was a graduate student
in the laboratory where I was
getting that Ph.D. And in short,
this is what the-- an
acetyl lysine surface looks
like of the histone.
So here it is.
Taking out here is an acetyl
and then here's trapoxin
which looks remarkably
like this lysine,
this acetyl lysine, right?
This look very, very similar.
Maybe a slightly
different number of carbons
but it looks very, very similar.
And so, this is going
to be a one possible way
to design inhibitors
of enzymes which is
to mimic the substrates, OK?
So this is the starting material
for the histone deacetylases,
the enzyme that chops
off this acetyl group.
And then this compound
over here is going
to inhibit that deacetylation.
There's more written
about this in the book.
But in any case, this two
look very similar and so,
that substrate mimicry,
that mimicry
of the starting material
is a very common way
to inhibit enzymes.
It works really well.
We're going to see that time
and again throughout this class.
OK, I went to change gears now.
I've shown you all
the cool things
that you can build out of DNA.
I want to talk to
you next about how
to actually synthesize the DNA
so you can build these things.
OK. If you want to make
happy faces or maybe you want
to make the first
frowny [phonetic] faces
out of DNA hasn't been done
before to my knowledge.
You're going to have to know
how to synthesize the DNA
so that you can make
that happen.
OK, so I'll first-- I'll talk
very briefly about DNA synthesis
in the lab but first I want to
talk to you about DNA synthesis
by the enzyme DNA polymerase.
OK, so DNA polymerase takes
a single-stranded piece
of DNA called the template
and adds a second strand
of DNA to that template.
OK. Now, all DNA
polymerase that's found
on the planet have
a common mechanism.
And they all require a starting
primer strand that gets the--
that gives it sort
of a running start.
Without this running start,
the enzyme doesn't know
where to begin and this is
actually a very useful property,
right?
You don't wan DNA
polymerase to come along
and start synthesizing random,
you know, bits of pieces
of DNA here and there.
And it turns out that this is
one that's been exploited quite
a bit.
And I'll show you some
examples of that in a moment.
OK, so this starter is called
a primer, the starting--
it forms again, double-stranded
DNA with the targeted template
and then DNA polymerase
lengthens this priming strand
in a five prime to
three prime direction.
In other words, it grabs
onto these three prime.
It adds the new five
prime, et cetera, OK?
So this direction here is
also common to all forms
of DNA polymerase
found on the planet,
five prime to three prime.
The starting materials here
are nucleotide triphosphates
structures-- so it's
a nucleotides
that I showed you earlier
but with triphosphates
attached to them.
OK. And basically what its doing
is again it's taking the green
primer strand and
lengthening it as shown
by this arrow over here.
So this is a classic
experiment that was done
that applied this principle
to crack the genetic code.
The genetic code is the
code by which sequences
of DNA spell out
amino acids, OK?
And this was back in
the 50s and early 60s.
There's this enormous
mystery about what
that code actually was.
OK. It was like this, you know,
unsolved major, major problem
and Marshall Nirenberg--
I think it's Rockefeller.
I might be wrong about that.
All right.
Marshall Nirenberg
used this property
to crack the genetic code.
What he did was he synthesized
templates that were long strings
of particular DNA sequences, OK?
So he made a long string of As
for example and then he looked
at not what was synthesized
by DNA polymerase
but downstream what
was made by ribosomes
when you give them
a long string of As.
And then by doing
that, he could figure
out what the genetic code was.
OK. So again, DNA
polymerase requires a template
to lengthen the existing strand.
Only RNA polymerase can
start from scratch, OK?
So RNA polymerase is kind of
an exception to this rule.
DNA polymerase requires
a priming strand reverse
transcriptase which takes
RNA and synthesizes DNA,
we saw that earlier
also requires a primer
and deoxynucleotide
triphosphates.
RNA polymerase is kind
of a special case.
By the way, any questions?
You guys feel free to interrupt
if there's anything
that comes up, OK?
Anything that's unclear, you
want to know more information
about it, don't hesitate
to stop me, OK?
Yeah.
>> Does the primer get
replicated as well or is it--
>> The primer gets extended
but it doesn't get replicated
in the load that
I'm showing you.
When we talk about PCR,
we'll show that it actually
can get replicated, so, OK.
That was a good question.
Other questions?
Yeah.
>> If you wanted to
hybridize [phonetic]
like a piece of a primer--
>> Yeah.
>> Lamination [phonetic] in it--
>> Yeah.
>> How many like base pairs
do you usually need to--
>> OK, these are
great questions.
OK. Awesome.
I'm glad you're asking.
I forgot to ask your names.
What was your name?
>> Paul [assumed spelling].
>> Paul and?
>> Anthony [assumed spelling].
>> Anthony.
OK. So Anthony's question
is how many base pairs
of DNA should you
have to get the--
to use as the primer to
get DNA polymerase going.
OK. And it kind of depends, OK?
So, you want DNA
polymerase to pick
up a specific gene and/or
a specific sequence of DNA
within a complex mixture.
And so, if you want to
pick up a specific gene
in the human genome,
you need a primer that's
at least 18 base
pairs in length.
OK, that's kind of
a magic number.
OK? So, 18 base pairs means that
you're uniquely encoding one
and only one gene in
the human genome, OK?
Thanks for asking.
It's a good question.
On the other hand, if you
want to do this at, you know,
a lower temperature, you can use
the Wallace rule and get away
with maybe a shorter sequence.
OK, maybe you don't
need such specificity.
Maybe your mixed-- your starting
population is less complex.
OK? Thanks for asking
Anthony and Paul.
OK. Let's move on.
OK, so here's a close up view
of what I've been telling you
about in hand waving examples.
We're now zooming down to the
level of atoms and bonds that,
of course, is what
really thrills me.
So, here is the primer and DNA
polymerase, not shown inside,
of course, this primer is
forming a double-stranded DNA
to the template strand
and also not shown is
that the template
strand must have adenine.
Over here, the hybridized to
the sliming [phonetic], OK?
In any case, the starting
material used here is a
deoxynucleoside triphosphate.
Note that deoxy at the
two prime hydroxyl.
OK. And here is the
two prime-- or sorry.
Here is the triphosphate
functionality.
The living group in
this reaction is going
to be diphosphate which
is use very commonly
in biology as a living group.
This is nature's tosylate
or mesylate that you learned
about back in Chem-51C.
This thing works really well
as a living group and it's one
of the reasons why we're going
to see it quite a bit
as a living group.
The-- All DNA primaries, all
enzymes that use diphosphate
as a living group absolutely
require a dication to bind
to this diphosphate and
their requirement is
for magnesium in DNA polymerase.
OK? So, actually a very
common problem that I see
in my own libratory when a
newbie shows up in the lab
and they have trouble
with their DNA polymerase,
nine times out of 10 it's
due the low concentrations
of magnesium.
OK? And there's a lot of ways
to get low concentrations
of magnesium.
So, a little tip.
OK, so here's magnesium.
What is it doing?
Magnesium is a Lewis acid that's
chelating to this diphosphate
and stabilizing its
negative charge.
Doing this makes it a
better living group, right?
This means that if it goes
out into the solution,
it doesn't require some
massive rearrangement of water,
it's already been stabilized.
It's at lower potential energy
then it would otherwise be.
OK, so here's the role
of DNA polymerase.
And I'll show you
instructionally what it looks
like it a moment.
DNA polymerase brings together
the three prime hydroxyl
of the priming--
the primer together
with this incoming nucleotide
triphosphate and then sets
up a nucleophilic
attack on the phosphorus
of the nucleotide triphosphate.
Note too that there is
a second magnesium ion
in the active site.
This second magnesium
ion does two things.
Number one, it helps to
stabilize the alkoxide formed
when this three prime
hydroxyl is deprotonated, OK?
So notice that it's forming
an ion pair relationship
with this alkoxide.
Number two, it actually
increases the nucleophilicity
of the lone pair that's going
to this nucleophilic
attack over here.
OK, so what magnesium is doing
here is it's making available--
better available this
long pair for an attack
by helping promote the
deprotonation of that hydroxide.
If the hydroxide
is deprotonated,
there's more long pair that's
available for the attack, right?
Make sense?
OK. This forms an intermediate.
There's a clasp [phonetic] of
intermediate and not only gets
to what we saw on Tuesday when
we looked at the hydrolysis
of DNA, exacts in mechanism.
That intermediate is depicted.
I'm showing arrows over here.
But again, we've looked at
that intermediate before
so I fell comfortable
living it off at this slide.
OK, any questions
about this mechanism?
Yeah.
>> Where does the
magnesium come from?
>> Great question,
what is your name?
>> Nick [assumed spelling].
>> Nick, OK.
Nick's question is where
does the magnesium come from?
Magnesium comes from
the food you eat.
It comes from, you know,
you added to the test tube.
So, typically we'll
add magnesium chloride
or magnesium sulfate directly
to the eppendorf tube.
Then you'll test tube that we
use for these reactions, OK?
But in humans reading,
you know all kinds
of food as magnesium in it.
OK. So, but it's
absolutely essential, OK?
So without the magnesium this
reaction does not go, OK.
It makes sense because I'm
showing you what a key role
it place.
OK, let's look structurally at
what this actually looks like?
This is an enzyme that
again has a number
of different orthologs
or homologs.
These are enzymes that do
more or less the same thing.
Reverse transcriptase
synthesizes DNA
from an RNA template.
The enzyme Taq is a DNA
polymerase that's use quite a
bit in research for
a tool called PCR,
which I'll talk about
in a moment.
But all of these enzymes have
a right handed structure, OK.
And here's what the
structure looks like.
OK, so here's a right
hand over here.
Here's my right hand.
And it's grabbing
on to the DNA, OK?
So the DNA is in red
and orange over here
and here is the enzyme
grabbing onto this DNA.
Now what happens is during the
synthesis, the DNA treads itself
through the crack form
by my thumb and palm, OK?
And as at a certain-- when the
crack nucleotide triphosphate
binds to the priming strand,
the newly synthesize strand,
the enzyme can then close.
When it closes, the palm and the
thumb get closer to each other,
palm, thumb and fingers.
OK. So it closes a
little bit like this.
Each time that closes,
that brings the magnesium's
up to the triphosphate
setting up formation
of the covalent bond
that I showed
on the previous slide, OK?
So each time the hand closes,
that's one nucleotide
that's been out.
OK, so let's do this, right.
We have this one bond.
OK, now let's do a couple
more, bond, bond, bond, OK?
Now here's the deal.
This enzyme is really cranking.
It's actually going to do
a thousand up to a thousand
of this per second for
some of these enzymes, OK.
So that's like, you know, too
fast for it to see really.
OK. So this enzyme can
really turn over very quickly
and actually this is
actually something
that my libratory is
directly observed.
We actually have watched one
of this enzyme cranking over
and we've watch differences
as we add difference
substrates to the enzyme.
It's really absolutely
fascinating series
of experiments.
OK, so, all of these enzymes
use a common mechanism.
Again, the enzyme doesn't close
until it gets the crack
nucleotide triphosphate
that binds to it at
that point it closes.
So in fact actually the
rate determining step
for this enzyme is
actually the rival
of the cracked nucleotide
triphosphate.
A is to Ts or DATP to Ts,
DCTP to Gs, et cetera.
OK, I'm going to tell you a
little bit more about why I'm
such in love with this enzyme.
This is a 3D machine.
I like fast cars and
I like fast enzymes.
This one is really amazing.
So check this out.
Imagine that double-stranded
DNA was about a meter or so
in diameter, OK, running a long
the length of this room, OK?
So we've got some DNA running
through the room, all right.
If that was true, DNA polymerase
would be about the size
of the FedEx delivery track.
OK. Including the polymerase
so there's some other
replication machinery
that I'm living off for
now that's involve as well.
But it would be the
roughly the size
of FedEx delivery track
pulling up right here, OK?
But here's the thing.
This delivery track
would be racing along
at about 375 miles per hour.
And that's how fast
DNA polymerase is going
in scale to the DNA.
Furthermore, it's making about
a thousand covalent bonds per
second which is insanely,
insanely fast.
And in addition, I
haven't talk about this yet
but there are other
subunits of DNA polymerase
that are providing
an error checking
and a correction function
such that the enzyme is making
to scale one error every a
hundred or six miles or so, hey,
which is extraordinary,
OK, 375 miles and hour
and one error every 106 miles.
OK, this is truly
remarkable stuff.
You could read more in
this reference down here.
OK, now, because this
enzyme is so efficient
and so superbly specific
at getting the right
Watson-Crick base pairing,
this has been used very
commonly in lots and lots
of laboratories, chem-bio
labs, molecular bio lab,
biochemistry labs,
forensic laboratories,
all kinds of labs used DNA
polymerase and they often use it
to amplify up copies of the DNA
using a technique called the
polymerase chain
reaction invented
by Kary Mullis [assumed
spelling] amongst others.
The way this works is you
start with some target sequence
of DNA shown here in purple.
Again, we'll call the
target B template, OK?
So that's the template DNA
that you're going to amplify.
Now, there are going to be three
steps to this PCR reaction.
In step one, we hit the DNA
up to high temperature
say 95 degrees.
And as we've discussed earlier
today, DNA when it's heated
up the high temperature goes
from double-stranded
to single-stranded.
It falls apart.
In step two, the
solution is cool down
and that allows the primers
shown here in green and blue
to a [inaudible], in
other words hybridized
to the single-stranded DNA.
Note that these two purple
strands don't find each other.
The concentration of
template if very, very low.
In fact, you can get down
to just a few copies of DNA.
So they never find each other.
They are like, you know,
lost from each other
after the heating step.
But you have a high
concentration of this green
and blue primers that can grab
on to the crack sequence of DNA.
That motion targets DNA
polymerase, drags DNA polymerase
to synthesize a specific
stretch of DNA.
And then that's done
in the third step
when the primers are extended
using again DNA polymerase,
DNTPs, magnesium chloride and
a temperature of 72 degrees.
To make this work, we use
a special DNA polymerase
that likes to run at 72 degrees.
It's a type of polymerase called
taq which is found in hot fence,
hot springs and it's in
an organism that's found
in this hot springs
that has evolved
to operate at this temperature.
And so, at 72 degrees, the
enzyme starts cranking.
At the lower temperature,
it's not working.
At the higher temperature,
it stops working.
But in 72 degrees, it's
loving [phonetic] life.
Again this is Celsius.
This is pretty warm, and its
start synthesizing this black
strand of DNA.
If you do this process a whole
bunch of times, each cycle,
you get a doubling
of the amount of DNA
and so you do this 30 times.
You get a huge amplification
for some target template of DNA.
Some target sequence of DNA.
OK. Makes sense?
Any questions about this?
I'm hoping I'm not telling you
anything you don't already know.
PCR is now taught like high
school and stuff like that now.
So, OK, summary.
Right hand role, we
looked at species
about the magnesium two plus,
stabilizing the nucleophile.
We've looked at this already.
Why don't we move on?
OK, so DNA polymerase is also a
terrific target for inhibitors
and reverse transcriptase
inhibitors have been very,
very important compounds for
stopping synthesis of DNA.
There're many reasons
why you'd want
to stop the synthesis of DNA.
To treat, for example cancer,
where cells are dividing
uncontrollably
if you can shutdown
the replication of DNA,
you have an effective
way of stopping cancer.
And in fact actually, childhood
leukemia's were stopped
in their tracks back
in the late 70s
through the wonderful
research of one
of my scientific heroes the
great Gertrude Elion shown here.
Gertrude Elion was born in 1918.
Her parents wanted her
to become a nurse, OK?
So they send her to
college and they said,
"Go and become a nurse."
She actually wanted
to become a chemist,
and when World War II broke out,
she was given her
opportunity, OK?
So during World War II, the man
were sent to the front to fight
and there are a lot
of opportunities
that were available to women
that weren't available before
that and she's one
of those people
who took that opportunity.
She joined Burroughs-Welcome
where she worked
with George Hitchings
for her entire career.
She's one of this people
who spend her entire
career at a single company.
And together with
George Hitchings,
she discovered this
class of compounds
that inhibits DNA
polymerase and ended
up after having a major
impact on childhood leukemia.
She is, you know, she is a
true superstar of science.
OK, one last thought.
Gertrude Elion never
received her Ph.D. She went
on to receive a Nobel Prize
in the 80s for this work.
She did it through
sheer force of will,
through her determination
to contribute something.
And I highly, highly
recommend in interview of her
and I'll leave and put it
up on the board over here.
There's-- If you want
to learn more about her,
this is terrific
interview of her that's
in the documentary
that I recommend.
OK, so the documentary
is called Isaac--
can you see this OK, normally?
OK. "Isaac-Newton and Me"
and the director is the
great Michael Apted.
I could actually have
a whole class just
on Michael Apted's
documentaries.
But in any case, she's
interviewed in this documentary
and she talks about
the incredible pride
and just the joy that she felt
when she would visit
children's hospitals
and she would see kids being
treated for the first time
with her compounds, and
how transformative that was
in the life of these kids.
These are kids that were,
you know, slated to diet
at very young age, just
like the ages of 10 and 12
and that were suddenly getting
cured by these compounds.
OK, let's take a closer look
and understand how
these compounds work.
OK, so she invented a series
of inhibitors of DNA polymerase
that look like this, OK?
So this is one called AZT.
It's also used very as a--
anti-HIV compound because it
inhibits reverse transcriptase.
It looks kind of
like a DNA base.
It has a deoxy at the 2'
prime hydroxyl but in place
of the 3' prime hydroxyl,
there is an azide, OK.
So what happens is this gets
taken up and phosphorylated
to give a triphosphate and
then DNA polymerase attempts
to use it as a substrate.
But what ends up happening is
instead of a 3' prime hydroxyl,
there's an azide here and
the azide caps the synthesis
of the new strand
of DNA preventing it
from being lengthened, OK.
So similarly, this is a ddC
another compound that's used
in the treatment of HIV and
also leukemia and instead
of a 3' prime hydroxyl,
it has a hydrogen there.
And so again, it gets
used by DNA polymerase
and the polymerase can no longer
lengthen the nascent strand
of DNA, OK, so both of these
shutdown DNA syntheses.
I'm simplifying things
a little bit.
There is another class compound,
non-nucleoside analogs.
These are nucleoside analogs
that are also used against HIV.
But that kind of
gives you a taste
for what Gertrude Elion did.
She had a major, major impact
in the fight against viruses
and in the fight
against leukemia.
And I actually had the pleasure
of meeting her once in life.
The day I was getting my
Ph.D. at my graduation,
she was receiving an
honorary Ph.D. from Harvard
and I just shook her hand.
That's about it.
I didn't have any profound
conversation with her
which is to my regret.
But a truly remarkable woman,
a true superstar of science.
I can not say enough about her.
I can have a whole
lecture about her.
Why don't we move on?
OK, so, that's DNA
synthesis in the cell--
oh, question over here.
Yeah.
>> So these nucleotides
that she synthesized,
how did they deliver
[inaudible] cancers cell--
>> Oh, this is such
a good question
and I'm so glad you asked.
What is your name?
>> Bobbin [assumed spelling].
>> Bobbin?
>> Yes.
>> OK. So, Bobbin's question
is how do you get the compounds
to the cancer cell.
What we're going to
see time and again is
that cancer cells are actively
eating up every little bit
of nutrient that
they could find.
They will just be devouring
stuff that's around them.
And so for this reason, they
and other dividing cells
will more preferentially take
up drugs like these that
are fed to the patient
that are injected
into the patient.
OK. So, the problem of course
is that there are other cells
in the body that
are also dividing
and that will unfortunately
take up this chemotherapeutics
and also end up dying because
their DNA synthesis will
be impaired.
So, OK. Great question.
Thanks for asking.
OK. So, I want to-- I
don't have very much to say
about chemical DNA synthesis.
I think it's an obviously
amazing topic.
This is one of those
areas of organic chemistry
that is a true triumph.
OK, so, we're going from
strength to strength today.
In DNA synthesis in the
laboratory has been so optimized
that we're at the point where
we get 99.9 percent yields
for reactions, OK?
This is really the
ultimate goal in the quest
to do organic synthesis.
And it was set in line by
the great Gobind Khorana
who won a Nobel Prize
but also Bob Letsinger
and Marvin Carruthers.
These guys deserve a Nobel Prize
because this really did kick off
a revolution in biotechnology
that was made possible by the
synthesis of DNA to make primers
which in turn allows PCR, which
in turn allows smiley faces
out of DNA and all those
other great discoveries
that we've been talking about.
OK. Now, I just want you to
scan this topic in the book.
Don't get too worked
up about it.
In the end, we have these
machines that looked like this
where you have a bunch
of bottles down here
that inject-- that you can use.
You could program a
computer up here to open--
to inject reagents
directly into a flow cells
that have the DNA sequence that
you're trying to synthesize.
So you do this using solid
support base synthesis
where did-- the nascent
strand of DNA is be--
is attached to some sort or
bead and you basically flow
in the reagents one
after another
and couple the correct
nucleotide directly
onto the DNA sequence.
It's a little more
complicated than that
but here's what you
need to know.
If you want to synthesize
any sequence
of DNA that's 150 base
pairs, 150 bases or less,
you get on the web and you call
up someone probably in Texas
or somewhere like that.
And there will be a whole
warehouse of machines like this
and you enter into
some form on this--
on their website exactly the
sequence of DNA that you want.
And that sequence will get
ported to a machine that looks
like this and there'll be
this warehouse just filled
with these machines.
And then there's a bunch of
technicians on roller blades
that are going to
be running around
and keeping the machines
fed with reagents.
You won't see any of
this because at the end,
you'll get a FedEx package
with your DNA sequence
probably in a couple of days.
Some of these I think are
even overnight, right?
So overnight, you're
going to get your sequence
of DNA perfectly cleaned up,
purified, delivered to you
at 95 percent or 99
percent purity depending
on how much you decide
to pay for it.
And, you know, it will
be perfect every time.
You won't even have to
think about the chemistry.
I love that.
That's the goal of
organic synthesis.
The goal of organic synthesis
to make this totally turn key
so that we can then
use this thing
to answer biological problems
which is what I want
to you about next.
OK, here's one example of
using this, an amazing ability
to do DNA synthesis to
address biological problems.
You can print sequences of
DNA on microscope slides.
OK. So here's a machine that's
nothing more fancy than I think
in this case it's an
ink jet printing device.
OK. And it's going
to be printing
out little oligonucleotide
one after another
on this identical
microscope slides.
So each square down here
is a microscope slide, OK.
And across the surface of
these microscope slides,
we're going to have a bunch
of different sequences of DNA
that have been printed down
onto specific spots, OK.
So what this is going
to do is this is going
to give us an array
of different sequences
of DNA each one capable
of hybridizing forming
Watson-Crick base pairing
to a different other
sequence of DNA or RNA.
OK. This is a technique
called the DNA microarray.
OK. So, here's the
way this works.
Let me just show you
what it looks like, OK?
So now I'm zooming in
on the microscope slide.
OK. So here's what
it looks like.
Each spot over here is a
different sequence of DNA
as you set up the
hybridization such that you end
up with a fluorescently
labeled sequence, OK.
So each-- again, each spot has
a different sequence of DNA.
If there is a complimentary
sequence in your sample,
then you will see
fluorescence, OK?
So, in green, that tells you
that your sample has
this particular sequence.
OK. And it's known exactly what
the sequence is in green that's
down there, OK, because you've
synthesized the compliment
to put it down right there.
OK, so-- I don't know.
Let's just say this is the
gene that confers resistance
to beta-lactam antibiotics.
Yes, you have that gene because
you're seeing green spots
right here.
In practice, this can be made
a lot more complicated, OK?
Let's imagine now that we have
two samples, one that we label
with red sequences and one that
we label with green sequences.
OK. This allows us
to compare all
of the different DNA
sequences present
or RNA sequences
present comparing them
against red versus green, OK?
And let's take a closer look.
OK, so each oligonucleotide
hybridizes
to a different mRNA transcript
where the one sample is labeled
in green and the second
sample is labeled in red.
OK, here is the way this works.
On sample one, you use
reverse transcriptase
to convert all these
RNA into DNA
and you add the green
fluorophore.
In sample two, you use reverse
transcriptase and label all
of those with a red
fluorophore, OK?
So sample one is green, sample
two is red and then you add both
of those to the DNA microarray.
The ones that are in green are
telling you, "Oh, that gene is
up regulated in those
types of cells."
The ones that are on red is
telling you it's up regulated
in the other types of
cells and where red
and green overlap
you see yellow, OK?
So, this allows you to compare
experiment versus control
and the possibilities
are endless.
For example, you can look at
cancer versus non-cancer cells,
virus infected versus normal,
G1 phase of the cell
cycle versus S-phase.
I'll explain these phases in a
moment, young cells versus old,
drug treated versus
no drug, et cetera.
And so in the end, you
get these massive arrays
where you get a huge
amount of data.
Each of these red spots
that could tell you,
"Oh, that's a gene.
That's up regulated," and say,
"Cancer cells are
virus infected cells."
And then the yellow ones,
those are ones you don't
have to worry about.
The green ones though, right,
because that's the same
in both types of cells.
The green ones however are ones
that are up regulated in the--
let's say, the non-virus
cells, OK down regulated
in the case of the cancer cells.
So you can see in one very
simple experiment relatively
simple, little complicated,
you get out enormous amount
of information about the
transcription activity
of an entire cell.
Again, this is all set in
place by DNA synthesis,
chemical DNA synthesis.
OK, so here's an example of
this, a specific example.
The example I'm going to use is
the small molecule FK-506 also
called tacrolimus.
This is a-- immunosuppressant
that was discovered
by the chemical--
the pharmaceutical company
Fujisawa hence the name FK has
an immunosuppressants.
It is given to patients who
had an organ transplanted, OK?
So, this was kicked off sort
of a revolution in the area
of liver transplantation
for example
when it first became
available in the 80s--
late 80s and early 90s.
OK, so if you give this
drug to your patients
who have just had
the liver transplant,
they will not reject the
new transplant of liver, OK,
because their immune
system is suppressed.
You can kind of see the
problem with that approach.
All right.
I mean the immune system
is suppressed that means
that if they get it cold,
they're going to
be on big trouble.
Setting that aside, OK, there's
other things that you can do.
Let's try to look at
what pathways are changed
when we feed cells
this compound.
In doing so, we could start to
identify the pathways associated
with the immuno response.
And so here's a classic
experiment done
by the company called
Rosetta Inpharmatics now Merck
pharmaceuticals and in this
experiment they have two kinds
of cells.
One kind of cells are green.
They were treated with
the green fluorophore
so the transcripts are treated
with the green fluorophore
and in the red-- so these
have no drug added to them
and on red, those
transcripts were treated
with the compound I showed
on the previous slide
called FK-506.
In yellow, there's a whole
bunch of different compounds.
Check this out.
In green over here, this
is a-- this is a protein--
this is gene that encodes a
protein that must be turned off
when the compound is added, OK?
Notice that it forms
a bright green spot.
Over here, here's one
that gets turned on.
It forms a red spot.
The yellow ones,
the orange ones,
let's not worry about
that so much.
OK. But you can imagine
doing all kinds
of analysis of the stuff.
The bioinformatics side of
these things, the computation
to analyze the stuff,
fascinating, OK,
and a really exciting area
of computer science research.
OK, does this make sense?
OK, so one experiment, you get
the whole pathway that's being
targeted by this drug.
OK and I'm not telling you
very about the pathway now.
Maybe we'll talk about it later.
Yeah. Question at the back.
[ Inaudible Question ]
OK, yeah. So, in practice
you have a laser that scans
across the surface
of this microarray
and then you have a CCD camera
that captures the
intensity of each spot.
OK, I'm hoping this
is blowing your mind
because it certainly blows mind.
OK, so now, I wanted to change
gears a little bit and talk
to you about how to analyze
DNA-- sequences of DNA.
Before I do that, I'm
looking at the time here.
I'm going to run all the way
up to the 10 before the hour.
But I just want to tell you I
have some good news for you,
a little weekend treat.
Thanks for bearing
with me of the quiz.
The midterm will only cover
through chapter three.
There'll be no chapter
four in the midterm, OK?
So when we come back on Tuesday,
we'll be talking more about--
don't get ready to go.
I'm not going to stop now but
I just want to let you know,
we'll be talking more about
DNA and then we'll go into RNA.
But the midterm next Thursday, a
week from today, will only cover
through chapter three, OK?
All right, let's get
back to our discussion.
I want to switch
gears very slightly.
Earlier I alluded to
agarose which is used
in those Petri dish plates
that I showed you earlier.
Agarose has the structure
down here.
It's isolated from
seaweed, and it is a polymer
that is tightly crosslet
[phonetic] to form a notch, OK.
You can actually form little
bricks form of the stuff
and then you can apply the DNA
to one end of the agarose gel.
So here's your little
brick called an agarose gel
and you add your DNA
to one end of this gel
and then you use
electrophoresis to push the DNA
through the polymer, OK.
We recall that DNA as negatively
charged so it will be attracted
to the positively
charged terminal
to possibly charge electrode,
the cathode at one end of the--
of your electrophoresis
apparatus.
OK, so again, you have
this wires coming out.
They're going back to some
source of electricity,
some power supply and that's
pushing the DNA through the gel.
The DNA will get
separated out then
on the basis of its size, OK?
So, bigger DNA is
going to get caught
up in this complicated network
over here whereas the little
pieces of DNA are going
to find it easier to flow
through these little pores.
OK. So in practice, what
this looks like is this.
I think I've already shown
you one of these agarose gels.
This is the top of the gel
where the big pieces are.
Here's the bottom of the gel
where the little pieces are.
Again, the big pieces
have not migrated as far
because they got stuck in the
inner stitches of the DNA.
Frequently, we add what's
called a DNA ladder to one lane
of the gel and that
provides basically a ruler
that tells us what sizes
of DNA we're looking at.
How is this visualized?
Why are these DNA pieces in
this color over here lit up?
What do we add to the gel?
>> Ethidium bromide
>> Yes, ethidium bromide.
Thank you.
Yeah. We've added a diethidium
bromide that concentrates
in the DNA and is fluorescent.
This works really well.
This tells us a lot
about DNA length
and a little bit
about its structure.
You can look at for
example supercoiling using
this technique.
OK, yeah. Here's the
ethidium bromide.
Here it is intercalated
into DNA and this
at practice is what
it really looks like.
It has this bright purple color.
In addition to separating on
gels, we very commonly separate
out DNA using capillary
electrophoresis.
And this is a much more
effective way of separating
out DNA that it differs
by a single base in size.
So you can take a large
number of sequences of DNA
and then separate them out such
that if you have, you know,
100 meers [phonetic], 99
meers, 98 meers, 97 meers, 96,
et cetera all neatly separated
out using this technique called
capillary electrophoresis.
It's the same electrophoresis
technique
with a different mobility
layer-- different mobility phase
but it's basically using
electrophoresis again
but in a capillary.
OK. Here's why this
is important.
You can use this
gel based separation
as a way of sequencing DNA.
If you had some way of breaking
it up into little pieces
where each piece
differs by one base pair.
In practice, what we do is--
and I will only tell you
about this one over here.
OK, this is the old fashion
way to do this in the--
using this actually we
use a acrylamide gels.
This is back when
I was a post doc.
We no longer do this.
No one in lab does it this way.
We all do it this way.
Here's the way this works.
OK, what we do is we add
a very low concentration
of dideoxy terminators.
This is exactly what a-- was
invented by Gertrude Elion
on the Gertrude Elion slide
that I showed you earlier.
This is missing, the
3' prime hydroxyl.
It has a hydrogen
in place over here
and these 3' prime terminators
missing this hydroxyl shutdown
DNA polymerase synthesis.
When they do, they also
carry along a fluorophore
that has a specific wavelength
associated with it, OK?
So you add a one
percent concentration
of the dideoxy C inhibitor
that has a green flurophore.
You add one percent that has
G and has a red fluorphore.
You add one percent that has
T and has a purple fluorophore
and you see where this is going.
So you have four
dideoxy terminators.
Each one with its
own fluorophore
and you set this fluorophores
up so there's no overlap
of the wavelengths or a
little overlap as you can get.
In the end, you separate out
the sequences using capillary
electrophoresis and what
you can do is actually read
out based upon the read,
green or blue status of each
of those dice that
you have a sequence
that says GAT, CTT,
GTT, et cetera.
In our practice, we actually,
you know, simply take the data,
feed it into a program and the
computer gives us the sequence
at the end.
OK, so this stuff is
massively automated.
OK, in fact, we're at the point
where our lab simply sends
out the sequences and
there's another lab.
It used to be on campus but now
it's actually-- where is it?
>> I think its San Diego.
>> It's here?
>> San Diego.
>> San Diego.
OK. Yeah. So it's in San Diego
that does all the sequencing
for us and it cost only a
few dollars per sequence.
OK. And that's-- that price
has dropped enormously.
OK, any questions about
anything we've seen today?
Yeah, Anthony?
>> Well, I don't know if you
said but I'm a little curious
about donor [inaudible]
stuff there where you--
>> Oh, yeah, yeah, yeah.
Let's put that off.
>> OK.
>> OK. Yeah.
Fluorescents [phonetic]
technology is interesting.
All right, last thing, you
can use this DNA technology
for all kinds of things.
It's used very extensively.
You can even use it to
program changes in organisms
like drosophila and you can
use this to test hypothesis.
Little hard to tell but
you see the extra eyeballs
that are growing out
of this organism.
Eyeball at the stuck,
eyeball over here, yeah.
So you could actually program--
you can insert sequences of DNA
into these organisms and
[inaudible] them to get turned
on at specific times and
use this some test whether
or not we understand
what specific genes do.
And this is actually
as fascinating story
that actually deals with
a heat shock protein.
OK, there's a couple of ways
of modifying DNA in organisms.
One way is to do it
randomly using compounds
like this one whose
mechanism we'll talk more
about on Tuesday.
So let's stop here.
When we come back next time,
we'll be talking
more about this.
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