PROFESSOR: And today I'm going to talk about DNA sequencing.
And I want to start by just sort of illustrating
an example of how knowing the DNA sequence can be helpful.
So you remember in the last lecture,
we talked about how one might identify
a gene through functional complementation.
And this process involved making a DNA library that
had different fragments of DNA cloned into different plasmids
and then involved finding the needle in the haystack where
you find the gene that can rescue a defect in a mutant
that you have.
So if this line that I'm drawing here is genomic DNA,
and it could be genomic DNA from,
let's say, a prototroph for LEU2, the leucine gene.
So this is from a prototroph.
Then you could cut up the DNA with EcoRI.
And if there is not a restriction site in this LEU2
gene, you get a fragment that contains the LEU2 gene.
And then you could clone this into some type of plasmid
that replicates in the organism that you're introducing
it and propagating it in.
And so that would allow you to then test whether or not
this piece of DNA that you have compliments a LEU2 auxotroph,
OK?
Now one thing I want to point out
is that because these EcoR1 sites, these sticky ends,
would recognize this EcoR1 one end or this EcoR1 end,
you can imagine that this gene--
if the gene reads this way to this way--
it could insert this way into the plasmid.
Or it could insert in the opposite direction.
So it could be inverted.
So this would have some sort of origin of replication
and some type of selectable marker.
But if you have the same restriction site
it can insert one way or the opposite way.
That's just one thing I wanted to point out.
Now let's say rather than leucine,
you're interested in cycling dependent kinase,
and you had a mutant end CDK and you had this sequence
of your yeast CDK gene.
Well, rather than having to dig through a whole library
of pieces of DNA for the CDK gene,
basically you're sort of fishing for that needle
in the haystack.
If you knew the sequence of the human genome,
you'd be able to identify similar genes by sequence
homology.
And you could then take a more direct approach,
where you take--
let's say you have a piece of human DNA
now, double stranded DNA, and it has the CDK gene.
You could take human DNA with this CDK gene.
And you have unique sequence around the CDK
gene, which would allow you to denature this DNA.
And if you denature the DNA, you'd
get two single strands of DNA.
And you could then design primers
that recognize unique sequences flanking the CDK gene.
So you could imagine you'd have a primer here
and a primer here.
And then you could use PCR to amplify specifically
CDK gene from, it could be the genome or from some library.
And then you get this fragment here, which includes CDK.
So knowing the sequence of the genome
would allow you to more rapidly go from maybe a gene
that you've identified as being important in one organism,
and find the human equivalent that might be doing something
similar in humans.
So this step here is basically PCR.
And let's say the CDK gene had restriction sites.
Let's see, we'll say restriction site K and A here.
Then if you have these restriction sites
in your fragment of DNA, you can then
digest or cut that piece of DNA with these restriction
endonucleases.
And then you'd get a fragment of CDK
that has K and A sticky ends.
We'll pretend that both of these have sticky ends.
And now you have unique sticky ends between K and A.
And you might have a vector that also has these two sites.
And you could digest this vector with these two enzymes.
And that would allow you to insert the specific gene
in this plasmid.
And if you have two unique sites,
because K only recognizes K here and A only recognizes A,
then it will ligate in.
But you can do it with a specific orientation
because you have two different restriction sites.
So I hope you all see how it's with one restriction
site versus two.
All right.
Now let's say you want to do something more complicated
than this.
Let's say rather than just identifying the gene that's
involved in cell division, you want to engineer a new gene,
in order to determine where this particular protein, CDK,
localizes in the cell.
So we have CDK, which could be from yeast or human,
it doesn't matter.
And you want to engineer a new protein, basically,
that you can see.
So remember Professor Imperiali introduced
green fluorescent protein earlier in the year.
And this green fluorescent protein
is from a gene from jellyfish.
So now we could, using what I've told you,
reconstruct or engineer a gene that
has DNA from three different organisms,
in order to make a CDK variant that we
are able to see in the cell.
So remember, a green fluorescent protein is like a beacon,
if it's attached to a protein.
If you shine blue light on it, it emits green light.
And so you can use a fluorescent microscope in order to see it.
In this case, let's say there's also another restriction site
here, R. And let's say you have a fragment of GFP that
has two restriction sites, A and R. You could then
cut this fragment and this fragment
with these restriction enzymes A and R.
And you could insert GFP at the C terminus of the CDK gene.
So you could go and have a gene that has CDK GFP inserted
inside a bacterial vector.
Now which one of these junction sites
do you think would be most sensitive in doing
this type of experiment?
So there are three junction sites.
There's this one, this one, and this one.
Which is the one you're probably going
to put the most thought into when
you're doing this experiment?
Yes, Miles.
AUDIENCE: The A?
ADAM MARTIN: The A site.
Miles is exactly right.
This one is going to be important.
And why did you choose that site?
AUDIENCE: Of the three sites, two
are half insert, half originals [INAUDIBLE]..
But at A, both sides of it are inserts.
So [INAUDIBLE] carefully.
ADAM MARTIN: And if you're trying
to make a fusion protein, what's going
to be an important quality of this?
Malik, DID you have a point?
AUDIENCE: Well, they try to [INAUDIBLE]
we'd have to make sure that the [INAUDIBLE]..
ADAM MARTIN: Excellent job.
So Malik just pointed out two really important things.
To make this a fusion protein, you
have two different open reading frames.
These two open reading frames have
to be in frame with each other.
So this junction here has to be in frame where
GFP is in frame with CDK, meaning
that you're reading the same triplet codons for GFP,
there in the same frame as CDK.
Also, you want to make sure there's no stop codon here.
Because if you had a stop codon here,
you're just going to make a CDK protein.
And then it's going to stop and then
you won't have it fused to GFP.
And you guys will work through more of these in the homework.
So you'll be able to get a sense of it.
So now for the remainder of this lecture
and also for Monday's lecture, I want
to go through a problem with you.
Basically, if you have a given disease that's heritable,
how might you go from knowing that disease
is heritable to finding out what gene
is responsible for that given disease?
And this is going to involve thinking
about different levels of resolution, in terms of maps.
So the highest resolution map you can have
for a genome is the sequence.
You can have the full nucleotide sequence of a genome.
And that's the highest possible resolution
because you have single nucleotide
resolution as to what every single base pair is.
But that's like knowing like your apartment
number and your street number and basically knowing
everything.
But starting out, you might want to know what continent it's on,
or what country is it in.
And so you first have to narrow down the possible locations
for a given disease gene.
And that will, at first, involve establishing what chromosome
and what region of a chromosome a given disease
allele is linked to.
And that involves making essentially a linkage map,
where you establish where a disease gene is located based
on its linkage to known markers that are present in the genome.
Now this is going to require that you remember back
two weeks ago, to when we talked about linkage
and recombination.
And you'll recall that we were looking
at the linkage between genes and flies and genes and yeast.
One difference between that type of linkage mapping
and human linkage mapping is we don't
have really clear traits that are defined by single genes.
You can't just take hair color and map the hair color gene
to link it to a disease gene.
Because hair color is determined by many, many different genes.
So in fruit flies, you can take white eyes
and see if it's connected with yellow body color
because both of those are determined by single genes.
So we need something other than just having phenotypic traits
that we can track.
We need what are known as molecular markers
to be able to perform linkage mapping.
And so what we need in these molecular markers--
well, if we just think about if we
wanted to determine the linkage between the A and B genes.
And if you did this cross, would you
be able to determine linkage?
Georgia, you made a motion that was correct.
Tell me.
Why did you shake your head no?
AUDIENCE: They'd all be heterozygous.
ADAM MARTIN: Yeah they'd all be heterozygous.
Because this individual has the same allele
on both chromosomes, you're not going
to be able to differentiate one chromosome from the other.
And so the point I want to make is
that in order to see linkage, what you need is variation.
So we need to have variation.
And another term for genetic variation is polymorphism.
So we need polymorphism, or genetic variation,
between these molecular markers.
We also need genetic variation in the disease.
But we have that.
We have individuals that are affected
by a disease and individuals that
are not affected by a disease.
So we have variation in alleles there.
But in order to map it with a molecular marker,
to map linkage to a molecular marker,
you also need variation here.
So the problem with this cross is here
you need to have heterozygote.
There needs to be variation in this individual, where
both of these alleles are heterozygous.
So now I want to talk about some of these molecular markers
that we can use, and how they vary between individuals
and between chromosomes.
Now this is going to be maybe the lowest resolution map.
But I'm talking about this linkage map here.
And you can see highlighted that the bottom here
are various types of polymorphisms
that we can use to link a given disease
allele to a specific chromosome and a specific place
on chromosome.
So I'll start with the first one, which is a simple sequence
repeat.
It goes by many names.
But I will stick with what's on the slide.
So a simple sequence repeat is also known as a microsatellite.
So you might see that term floating around,
if you're reading about this.
And what a simple sequence repeat is, as the name implies,
it's a simple sequence.
It could be a dinucleotide, like CA.
And it's just a dinucleotide that's
repeated over and over again.
So on a chromosome, you might have a unique sequence,
which I'll just draw as a line. ,
And then you could have a CA dinucleotide that's repeated
some number of times, N. And then that's followed by another
unique sequence.
And that's what's present in it.
So that would be one strand.
And then in the opposite strand, you'd
have a unique sequence, the complement of CA,
which is GT, and then, again, unique sequence.
And so there's variation in the number of repeats of the CA.
And so there's polymorphism.
So we can use this to establish linkage
between this marker and a phenotype,
like a disease phenotype.
So how might you detect the number of repeats
that are present here?
Anyone have an idea of a tool that we've discussed
that could be used here?
So one hint that I gave you is that the sequence here
is unique and the sequence here is unique.
So is there a way we can leverage that unique sequence
to determine whether there's a difference
in the number of repeats?
What's a technique we discussed that involves
some component of the technique recognizing a unique sequence?
Yeah, Natalie?
AUDIENCE: CRISPR Cas9.
ADAM MARTIN: Well, CRISPR Cas9 is a possibility.
Jeremy, did you have an idea?
AUDIENCE: PCR?
ADAM MARTIN: PCR-- so it's true.
You could get it to recognize that.
But then you have to detect it, somehow.
So what's more commonly used is PCR.
Those are both good ideas.
But using PCR, you could design a primer here
and a primer here.
And you could amplify this repeat sequence.
And the number of repeats would determine
the size of your PCR fragment.
So if you did PCR, then you'd get a PCR fragment that
has the primers on each end, but then has this certain size
based on the number of repeats.
So in that case, we need some sort of tool
that enables us to determine the size of a particular DNA
fragment.
And so I'm going to just introduce to you one such tool,
which is gel electrophoresis.
And gel electrophoresis involves taking DNA
that you've generated, by either PCR or by cutting up
DNA with a restriction enzyme, and loading it
in a gel that has agarose.
Maybe it's composed of agarose.
It could be composed of polyacrylamide.
And then because DNA is negatively charged,
the backbone, if you run a current through it,
such as the positive electrode is at the bottom,
then the DNA is going to snake through this gel.
Now we'll do a quick demonstration,
if you two could come up.
I need one volunteer.
Ori, find 10 of your friends and bring them down.
All right.
That's probably good.
Yeah.
All right, Hannah, why don't you--
you guys have to link up, OK?
Stay over here.
We'll start at this end.
This is the negative electrode over here.
The positive electrode is going to be down there.
And Jackie is going to be our single nucleotide.
You guys link like--
yeah.
You don't have to do-si-do, or anything like that.
All right.
Now what I want you guys to do is
I want you to slalom through these cones
like it's all agarose gel.
So that you're going towards the other side.
And I'm going to turn on the current now.
So go.
All right, stop.
All right.
See how the shorter DNA fragment is
able to more easily navigate through the cones
and get farther.
So it was somewhat rigged.
I know.
But I just needed some way to make sure you always
remember that the shorter nucleotide, or the shorter
fragment, is going to migrate faster.
You guys can go back up.
Thank you for your participation.
Let's give them round of applause.
[APPLAUSE]
All right.
So what you just saw is that the longer DNA fragments,
they're going to be more inhibited by moving
through the gel.
And so they're going to move slower and thus,
not move as far in the gel.
Whereas, the small fragments are going
to move much faster because they're
able to maneuver their way through this gel
much more quickly.
So there's going to be an inverse proportionality
between the size of the DNA chain and its rate of movement.
You're always going to see the shorter DNA
fragment moving faster.
So what one of these gels actually
looks like is shown here.
So this is a DNA gel that's agarose.
And DNA has been run in these different samples.
And what you're seeing is this gel
is subsequently stained with a dye,
like ethidium bromide, which allows you to visualize
the individual DNA fragments.
And so a band on this gel indicates a whole bunch
of DNA fragments that are all roughly the same length.
So essentially, you can measure DNA length
using this technique.
What's over here at the end of the gel,
this is probably some sort of DNA ladder,
where you have DNA fragments of known length
that you can use to calibrate the length
of these bands over here.
So this is how you measure DNA length.
And we're going to use it over and over again,
as we talk about DNA and sequencing.
So now, let's think about how this
is going to help us establish linkage
between a particular marker in the genome
and a genetic disease.
So if we think about these microsatellite repeats,
I told you they're polymorphic.
They exhibit a lot of variation in size.
And so here's an example showing you
a female who has two intermediate sized
microsatellites.
And if you look at this--
if you did PCR and measured the size of these,
you get two different bands because there
are two different alleles of different length here.
So you can see this individual has two intermediate length
repeats.
And this person has had children with an individual
that has a short and a long microsatellite.
And you can see that on the gel, here.
Now this female is affected by some disease.
And these two individuals have children.
And you can see that a number of those children
are affected by the disease.
So what mode of inheritance does this look like?
If you had your choice between autosomal recessive,
autosomal dominant, sex linked dominant,
and sex linked recessive, what mode of inheritance
is this looking like?
Oh, Carmen.
AUDIENCE: Autosomal recessive.
ADAM MARTIN: Autosomal recessive?
Why do you go with recessive?
Yeah, go ahead.
AUDIENCE: Because there is a male that's affected.
But not both of the parents are affected.
So it seems like the father is heterozygous
and the mother is homozygous recessive.
ADAM MARTIN: That's possible.
That's exactly the logic I want to see.
Is there another possibility?
Yeah, Jeremy.
AUDIENCE: Autosomal dominant.
ADAM MARTIN: It could also be autosomal dominant.
So you're right.
You're right.
If this was not a rare disease, then that male
could care be a carrier and could be passing it
on to half the children.
So that's good.
You'd essentially need more information
to differentiate between autosomal recessive
and autosomal dominant.
For the purposes of this, we're going
to go with autosomal dominant.
And what you see is that you want
to look at the affected individuals
and see if the disease phenotype is linked, or connected,
with one of these microsatellite alleles.
So if we look at--
we basically PCR DNA from all these individuals.
And if you look at who is affected,
each one of the individuals has this M double prime band.
And none of the unaffected individuals has it.
So obviously, it would be better to have more pedigrees and more
data to really establish significance
between this linkage.
But this is just a simple example,
showing what you could possibly see
if you have one of these molecular markers linked
to a particular disease allele.
So that kind of establishes the principle.
Now let's think about what are some other molecular markers
that are possible?
So another type of marker, and this
is one that's the most common one, if I go here.
So here, you see here's is a linkage map, here.
And you see most of these bands are green.
And the green markers, here, are what
are known as Single Nucleotide Polymorphisms, or SNPs.
So single nucleotide polymorphisms--
and this is abbreviated SNP.
And what a single nucleotide polymorphism is, is it's
a variation of a nucleotide at a single position in the genome.
So it's just a one base pair difference at a position.
So there's variation of single nucleotide at a given position,
at a position in the genome.
And because that's a pretty general definition,
there are tons of these in the genome.
Now one thing to think about is you
could have a mutation in an individual that creates a SNP.
So you could have a de novo formation of a SNP.
But if you have a SNP and it gets
incorporated to the gametes of an individual,
then that variant is going to be passed
on to the next generation.
So this is something that could occur de novo.
But it is also heritable.
And if it's heritable, then you can follow it
and use it to determine if a given
variant is linked to a given phenotype, like a disease.
So to identify a single nucleotide polymorphism,
it's helpful to be able to sequence the DNA.
And I'll talk about how we could do that in just a minute.
But before I go on, I just want to point out
a subclass of SNPs that can be visualized without sequencing.
And these are called restriction fragment length polymorphisms.
So restriction fragment-- so it's
going to involve some type of restriction
enzyme digest length polymorphism.
It's a long word.
But it's abbreviated RFLP.
And what this is, is it's a variation
of a single nucleotide.
But this is a subclass of SNP.
Because this is when the variation occurs
in a restriction site for a restriction enzyme.
So if you remember your good friend EcoR1,
EcoR1 recognizes the nucleotide sequence GAATTC.
And EcoR1 only cleaves DNA sequence that has GAATTC.
So if there was a single nucleotide variation
in the sequence, such that it's now
GATTTC, or something like that, that destroys the EcoR1 site.
And so EcoR1 will no longer be able to recognize this site
in the genome and cut it.
So you could imagine that if you had
one individual in the genome having three EcoR1 sites,
if you digest this region, you'd get two fragments.
But if you destroyed the one in the middle,
then if you digested this piece of DNA,
then you'd only get one fragment.
And that's something.
Because it results in different sizes of fragments,
that's something you can see just
by doing DNA electrophoresis.
And maybe you would use some method
to detect this specific region, so that you're not
looking at all the DNA in the genome,
but you're establishing linkage to this specific area.
You could use PCR.
You can have PCR primers here and here.
And you could then cut with EcoR1.
In one case, you'd get two fragments.
In this case, you'd get two fragments.
In this case, if you amplified this region of the genome
and cut with EcoR1, you'd only get one fragment.
So you'd be able to differentiate
between those possibilities.
Yes, Malik.
AUDIENCE: When you use PCR, are there [INAUDIBLE]??
ADAM MARTIN: What's that?
AUDIENCE: Are there [INAUDIBLE]?
ADAM MARTIN: Oh.
You're saying what causes it to stop?
That's a great question, Malik.
Yeah.
So initially, it's not going to stop.
That's absolutely right.
But because every step, each time you replicate,
it's then primed with another primer.
So you'd replicate something like this that's too long.
But then the reverse primer would replicate like this.
And it would stop.
So if you go back to my slide from last lecture,
look through that and see if it makes sense how it's ending.
Because if you do this 30 times, you really
will enrich for a fragment that stops and ends at the two
primers, or begins and ends at the two primers, I should say.
Good question.
Thank you.
All right.
Now, let's talk about DNA sequencing.
Because as I showed you, obviously,
these SNPs, because there are so many of them,
are probably the most useful of these markers to narrow in
on where your disease gene is.
And to detect a SNP, we need to be able to sequence DNA.
So I'm going to start with an older
method for DNA sequencing, which conceptually,
is very similar to how we do DNA sequencing today.
And so it will illustrate my point.
And then at the end, I'll talk about more modern techniques
to sequencing.
So the technique I'm going to introduce to you
is called Sanger sequencing.
And that's because it was identified by an individual
named Fred Sanger.
And I'm going to just take a very simple DNA sequence,
in order to illustrate how Sanger sequencing works.
So let's take a sequence that's really simple.
This is very, very simple, and then more sequence here.
So let's say we want to determine the nucleotide that's
at every position of this DNA fragment.
So one way we could maybe conceptually
think about doing this, is to try
to let DNA polymerase tell us where given nucleotides are.
And if we're going to use DNA polymerase, what
are we going to need, in order to facilitate this process?
Yes, Rachel.
AUDIENCE: [INAUDIBLE].
ADAM MARTIN: You're going to need nucleotides, definitely.
So we're going to need nucleotides.
What else?
To start, what are you going to need?
Miles?
AUDIENCE: Primer.
ADAM MARTIN: You're going to need a primer, exactly.
Good job.
So you need a primer.
So here's a primer.
And now, we're going to try to get DNA polymerase to tell us
whenever there is a given nucleotide in this DNA
sequence.
And so think with me.
Let's say we were able to get DNA polymerase to stop whenever
there was a certain nucleotide.
So if we go through just a couple nucleotides,
let's say, at first, we want DNA polymerase to stop whenever
there's an A. So let's say there was
a possibility it would stop at this A. If it's
stopped at this A, you'd generate
a fragment of this length.
But if it read on through that A,
there's another possibility that it would stop at this A.
So we're kind of looking at when these are stopping.
And the final possibility is it goes on and stops at this A.
So if this DNA polymerase stopped only at As,
you'd get fragments that are these three discrete lengths.
Now let's consider another possibility.
So pink here is stop at A. And in blue, I'm
going to draw what would happen if it stopped at T.
So they all start from the same place.
If it stopped at T, it would just
stop one nucleotide beyond this A in this simple sequence.
So in blue here, this is stop at T.
But if it's just a possibility, it stops.
And some of the polymerases could go beyond this T
and go to the next T and stop here.
And again, this would be one nucleotide length longer
than this pink one, here.
And the final one would--
I'll just draw it down here--
would get out to this last T, here.
So what you see is if we could get DNA polymerase to stop
at these discrete positions, we'd
get a different sized fragments, whether it
was stopping at one nucleotide versus the other nucleotide.
You all see how this is resulting in different fragment
lengths.
Yes, Andrew.
AUDIENCE: How would you create a pattern [INAUDIBLE]??
ADAM MARTIN: There are companies now.
You can basically take nucleotides
and synthesize these primers chemically,
not using DNA polymerase.
AUDIENCE: I'm saying how would you know what primer to use,
if you don't know the sequence?
ADAM MARTIN: Oh, in this case, you'd
have to start with some sequence that you know.
So in most sequencing technologies,
you kind of make a DNA library, where you know
the sequence of the vector.
And then you'd use the vector sequence as a primer
to sequence into the unknown sequence.
Great question.
Good job.
All right.
So what we need now then is some sort of tool or ability
to stop DNA polymerase when there's a certain nucleotide
base.
And to do that, we can use this type of molecule,
here, which is known as a dideoxynucleotide.
Remember, for DNA polymerase to elongate a chain,
it requires that the last base have a three prime hydroxyl.
And so what this dideoxynucleoside triphosphate
is, is it's a nucleoside triphosphate that
lacks a three prime hydroxyl.
Here, I'll highlight that.
So you see this guy?
You see it bolt the highlight H?
There's a hydrogen there on the three prime carbon,
rather than the normal hydroxyl group.
So if this base gets incorporated into a elongating
chain, DNA polymerase is not going to be able to move on.
So this method where you can add a certain dideoxynucleoside
triphosphate to stop chain elongation
is known as a chain termination method.
So you're getting chain termination.
And you're getting this chain termination
with a specific dideoxynucleoside triphosphate.
So these dideoxynucleotide triphosphates,
if they get incorporated into the DNA,
are going to halt the synthesis of that DNA strand.
So if we take our example, here, this
might be a reaction that has dideoxythymidine triphosphate.
So if we had dideoxythymidine triphosphate in this sample
and it's elongating, then when the polymerase reaches
this point, there's a possibility
that it will incorporate the dideoxynucleoside triphosphate.
And if this is a dideoxynucleoside triphosphate,
then there won't be a three prime hydroxyl.
And DNA polymerase will just be like, oh, I can't go on!
Because it's not going to have a three prime hydroxyl.
So it's not going to be able to continue
with the next nucleotide.
So this is known as chain termination.
So let me take you through an example, here.
All right.
So here's an example that you have a slide of.
And again, there's a template strand,
which is the top strand.
And this method requires that you have a primer.
And what's often done is you label the primer.
So the first step is you have to denature your DNA.
So you have to go from double stranded DNA
to a single stranded DNA.
And then you mix the double stranded DNA
with first, this labeled primer, such
that the primer can then yield to the single stranded DNA.
You need DNA polymerase, as I've mentioned.
And as, I believe, Rachel mentioned
before, you need the building blocks of DNA.
So you need the four dideoxynucleoside
triphosphates.
So you always have the four dideoxynucleotide
triphosphates.
But what's special here is you're
going to spike several reactions with one
of the dideoxynucleoside triphosphates.
So you spike the reaction with a tiny amount
of one of your dideoxynucleoside triphosphates.
So let's say you have a reaction, here.
And this this one here has dideoxyadenosine triphosphate.
Then polymerase will along get this strand
until there's a thymidine on the template.
And then there's a possibility that it will
incorporate this dideoxy NTP.
And if it does, then you get chain termination.
And you get a fragment of this length.
But the other possibility, because there is still
the deoxy form of the NTP present,
it's possible that it incorporates
a deoxyadenosine triphosphate there.
And keeps going, and then incorporates a dideoxy ATP
later on, where you have another T.
And so the polymerase will essentially randomly
stop at these different thymidine residues,
depending on whether or not a dideoxynucleoside triphosphate
is incorporated.
And that means for a given reaction, one in which
you have dideoxy ATP, you get a certain pattern
of bands that represent the length of fragments,
where you have, in this case, a thymidine base.
And then you do this for all four bases, where
you have four reactions, each with a different base that's
dideoxy.
So when you're adding these, you're
going to do four reactions, one with dideoxy ATP spiked in,
one with dideoxy TTP, one with dideoxy CTP,
and the last with dideoxy GTP.
And because these nucleotides are
present in different positions along the sequence,
you're going to get distinct banding pattern for each
of these reactions.
But using that banding pattern, you
can then read off the sequence of DNA that's
present on the template strand.
So this is how sequencing was done for many, many years.
These days, it's been made cheaper and faster.
And now what's often used is next generation sequencing.
And one the pain in the ass about sequencing before
is you'd use a lot of radioactivity.
Your primer would be radioactive,
so that you could detect these bands.
Right now, everything's done using fluorescence, which
makes it much nicer, I think.
And so in next generation sequencing,
your template DNA is attached to a solid substrate, such
that it's immobilized on some type of substrate.
And then you add each of the four nucleoside triphosphates.
In this case, they're labeled with a dye, such
that each one is a different color.
But the dye also functions to prevent elongation,
such that, again, it's this chain termination.
When you incorporate one of these,
the polymerase just can't run along the DNA.
It incorporates one and then stops.
So if you get your first nucleotide incorporated,
it will incorporate one of these four.
And it will be fluorescent at a certain wavelength, which
you can see using a device or microscope.
And then what you then do is chemically modify this base,
such that you remove the dye and allow it
to extend one more base pair.
And so you go one nucleotide at a time.
And you read out the pattern of fluorescence that appears.
And that gives you the sequence of DNA
on this molecule that's stuck to your substrate.
And you can do this in parallel.
You can have tons, many different strands of DNA.
And you can be reading out the sequence of each one
of these strands in parallel.
Great.
Any questions about DNA sequencing?
OK.
Very good.
I will see you on Monday.
Have a great weekend.