Good morning. Good morning.
I don't know about you, but I can't take too many more nights like this.
I confess, I haven't gotten a thing done for so many nights in a row now,
but what a game! How many of you saw the game? Excellent.
Very good, very good. You have your priorities straight in
the world. Very good. Well, if it's possible to get your
minds off Curt Schilling last night, and off more importantly tonight.
Perhaps we can spend a bit of time this morning in the meanwhile with
whatever spare neurons you have talking about recombinant DNA for a
bit, OK? What we talked about last time was different ways to clone
your gene based on its properties. We started off with cloning by
complementation, right, the idea that if you took a
library of clones, you would be able to put it into
bacteria and select a bacterium whose phenotype had been restored by
virtue of having the plasmid. You would complement the defect.
You'd find the clone you wanted because it complemented the defect.
That's great if you can put it into an organism that has a defect.
You can do it with bacteria. You can do that with yeast.
It's harder to do with large organisms because you can't inject
enough of them with different clones to be able to make that practical
unless you're working in cell culture or some very small,
fast growing organism. We talked about being able to use a
protein sequence, reverse translating that protein
sequence in the computer from amino acid sequence to nucleotide sequence,
and using the nucleotide sequence to design a probe to hybridize back to
the genome. That works fine if you have a protein sequence.
But the last topic we talked about that I wanted to just touch on again
this morning was suppose you were trying to clone the gene that causes
a certain human disease, and you have no idea what the
protein was. Then, you can't use its amino acid
sequence because you don't have the protein. What can you possibly do
when all you know is that you have a gene which causes a genetic defect
that causes a disease? And I said you could clone it using
the ideas of genetic mapping, position, the things that Sturtevant
developed. And, I touched on it briefly,
and I want to just touch on it a bit more because some people had some
questions about it. And I've set up a very simple
example to show you. Suppose that, to make it easy,
we're working in a fruit fly first. We're working drosophila, and
suppose that the true picture of the underlying chromosome is like this.
There's a locus that could either have a mutant allele M or the wild
type allele plus. There's a bunch of other loci along
the chromosome. And, let's suppose we know all of
where they are and all that. And, they have two alternative
alleles. At this locus the alleles are orange
or pink. At this locus I'll call the alleles orange or pink.
Now, these are different loci. These are different alleles. I've
just called them orange and pink in both cases so I don't have a rainbow
of colors up here to confuse us. But all I mean is there's two
possible alleles here, two alleles here, two alleles here.
This is the diseased gene we're interested in,
and these are passive markers. These are other markers along the
chromosome. If we were to set up a cross between
heterozygotes, a heterozygote here,
and a heterozygote here, and it were the case that on the
chromosome bearing the mutant allele, it happened that at these three
markers we had orange alleles. I don't know what they are, but
whatever these orange alleles are, they might be a visible phenotype,
forked or yellow or bristled. They could be a DNA sequence
difference. They could be whatever you want, but let's suppose the M
chromosome has a set of alleles that are different in each location than
the plus chromosome. Then, when we look at the offspring
that come out of this cross, let's only, for the sake of
simplicity, look at those offspring who are homozygous mutants.
Well, in general, if there's been no crossover here,
then the M chromosome will have orange, orange,
orange, orange, orange, orange. If there's been a crossover,
however, it could go orange, orange, pink on one of those
chromosomes. Or if there's been crossovers like this,
it could go orange, orange, pink on one chromosome, and orange,
pink, pink on the other chromosome. It could even,
in the extreme, have had crossovers very close to
the gene maybe here, and even maybe here. And you've got
orange, pink, pink, and pink, pink, pink.
But if we look at the many segregates, you know from genetic
mapping that the closer the locus is to the disease gene,
the more strongly correlated the inheritance will be,
the tighter the linkage will be. This is nothing more than linkage
mapping. But now, suppose we were doing
linkage mapping, but for the sake of argument the
whole genome had already been sequenced. Suppose the genome had
been sequenced in a cross, and the whole genome of the fruit
fly had been sequenced which it has been sequenced.
And, we looked at a cross and we looked at the mutants.
And what we did was we tried different positions along the genome.
And at each position, we had some genetic marker.
And that genetic marker might be as simple as the fact that at that
position, maybe there is an A in the DNA sequence on one of the
chromosomes, and maybe I don't know a G in the other sequence.
And over here, this marker might be, there's a T in some particular
position, and there's a C in some particular position.
If we could assay that, if we could tell, we could look
whether this spelling variation is closely correlated with the mutant.
And this spelling variation is closely correlated with the
inheritance of the mutant allele. And we could just try up and down
the genome, different sites of spelling difference as if they were
genetic markers in our cross because they are genetic markers in our
cross, and see which one is most tightly correlated.
The minute we get any genetic sequence difference,
that shows co-inheritance linkage in this cross, we know that this spot
in the genome must be nearby our mutation.
So, we'll try one closer, and we'll try one on the other side.
And, what you do is you test sites of genetic variation,
first to find one that shows any co-inheritance.
And once you've got that, you try ones closer, and closer,
and closer. Last time I talked about the process of,
if you had one of those markers you could use it to isolate the next
clone and the next clone and the next clone. But you know what I
realized? That's so old fashioned. We might as well deal with the fact
we have a sequence of the genome. No more would you ever isolate the
next clone and the next clone and the next clone.
You just look it up in the computer. So, even if you have the whole
sequence of the genome, we have to figure out what part of
it was co-inherited along with this disease, and that's the way you do
it, OK? Genetic mapping, ust as Sturtevant invented it,
can be applied if you have a whole sequence of the genome,
and enough sites of variation. And, I've drawn it for a fruit fly
cross, but this could equally well be cystic fibrosis.
The only difference is if we're doing this in human families and
it's cystic fibrosis we don't have as many offspring.
So, we have to pool data from many families. And,
we can't arrange it so that every family has exactly the same orange
alleles up here and pink alleles down there, but computers can deal
with that. They can still figure out the correlation across many
families, and you find the spot in the genome where for many,
many, many families the kids who all got the disease show correlated
inheritance with this marker. And that eventually pins you down
to a region of the genome. It pins you down to those genetic
markers that show the absolute tightest correlation,
tight correlation, and that's where you look.
And in that fashion, people went being able to map the
location of Huntington's Disease in 1984 to, by now,
mapping the locations of more than 1, 00 different human genetic diseases
where people didn't know the protein in advance. They did it entirely
based on this positional mapping. So, Sturtevant's idea, which I like
so much, has played itself out so beautifully now in the area of
modern molecular medicine. OK. So, onward.
I want to talk about a few other variations on the theme rather
quickly, and then I think I want to talk about how you analyze your
clones. First, variations on cloning,
I should just at least mention it. We talked about cloning in an
autonomously replicating plasmid in a bacteria.
So, you go to a bacteria. They have some autonomously
replicated pieces of DNA. There are circles. You can clone
in them, and you can typically, these things are on the order of,
I don't know, 1,000 to 2,000 to 5, 00 bases can be readily cloned in
these plasmids. You can do more,
but that's a typical kind of number is the insert size,
typically. But we in the lab go up to much higher numbers
like 10,000 sometimes. You can also, if you wanted to study
yeast, it turns out yeast happily have plasmids as well,
and you can do a similar sort of thing for yeast.
It turns out that instead of using plasmids, you can use bacterial
viruses. These bacterial viruses have all different shapes as we've
talked about, circular or linear, and they can typically hold, oh, 15,
00-40,000. Some of these viruses are quite big.
The bacteriophage lambda tends to carry a lot of stuff.
And, it can replicate. So, you could do the same thing to
that. You can even use viruses that infect mammalian cells and there are
all sorts of viruses now that people clone in again,
linear or circular. I don't know, for mammalian cells,
you often, the viruses like 1,000-5, 00. You can even make artificial
whole chromosomes now. You can do this in yeast.
Artificial chromosomes are called YACs. They have all the little
machinery, little telomeres on them, little centromeres. They have a
selectable marker, and then you can clone into it your
piece of DNA. And these can take up to a million bases of DNA.
So, if you wanted, there are bacterial artificial chromosomes.
They're called BACs if they're in bacteria. And recently,
people have developed artificial chromosome systems for mammalian
cells, and specifically human cells. And they're called unfortunately
MACs and HACs and things like that. Basically, any molecule that can
replicate in any system, some smart molecular biologist will
come along and say, how do I use that for my purpose,
to stick my DNA in it, and get it to replicate in this organism?
And so, if something's not on this list, it will be soon,
OK? Now, here's another thing. This is cloning chunks of DNA.
Just to have the piece of DNA in a library, but suppose we want to do
more than just have the DNA sitting there in the bacterium,
suppose what I'd really like to do is take a bacterium,
E coli, and put it to work for us. Maybe what I'd like to do is take a
plasmid and insert in that plasmid the gene for human insulin.
So, I'm going to take the DNA locus corresponding to human insulin,
clone it into my plasmid. Maybe I'll have isolated it from my
library because, let's see, insulin's protein
sequence is known so I could reverse translate it to a nucleotide
sequence. So, I could probe a library.
So, I could find the clone that has insulin. Now what I'd like to do is
persuade this bacteria not just to carry the DNA but to make insulin
for me. Would that be useful? Yeah, how did people used to get
insulin? Cadavers, dead bodies; it would be much easier
to get them from a fermenter, right, to get insulin from a
fermenter, if you could just ask E coli to make it.
So, if we put it into E coli, will it make insulin for us?
Here's the human locus, DNA for insulin. Will it make
insulin? Let's see, how do you make a protein?
You've got to start by making RNA, right? You've got to transcribe the
gene. Will E coli transcribe this gene?
Well, why? It's got a promoter, right? It's got the insulin
promoter. There we go. The insulin promoter is here.
So, E coli will come along to the insulin promoter and start making
RNA? No, it turns out that promoters in humans and promoters in
bacteria are sufficiently different. They don't work across species.
They won't recognize the human promoter. Too bad. Any ideas?
Yep? Stick a bacterial promoter there. Good, you're acting like a
good molecular biology designer here. Let's put a bacterial promoter here.
It will recognize its own promoter. That's great. Then, let's put the
DNA for the human insulin gene here. And now, maybe we'll put the Lac
operon, and when it has lactose it'll start making RNA from the
human insulin gene. And it'll start translating it.
And, we get insulin. Any problems? Well,
will it make any, for starters? What's another aspect of mammalian
genes that's different from bacterial genes?
Processing, what kind of processing with the RNA? And the splicing,
ooh, the insulin gene has introns that have to be spliced out.
So, this is going to make some RNA, insulin RNA, and it needs to be
processed like this. Will bacteria carry on our splicing
for us? They don't do splicing. Yep? Well, that's a very
interesting question because we haven't. But,
what do you propose? You see, I've just taken a piece of
human DNA from the human genome, which encodes the introns and the
exons. But, you seem to have a solution to our problem,
and what would that be? So, instead of making a library of
genomic DNA, what you're suggesting is a radical idea.
Let's instead take human RNA. Here's some human RNA, lots of
human RNA, a big collection of human RNA. What was at the end of the
human RNA: a poly(A) tail. And what I understand you to be
suggesting is if we take human mRNAs, a whole collection of them,
you want me to turn these mRNAs back into DNA and clone them instead of
using the chromosomal DNA. How do I turn an RNA back to DNA?
Is that possible? What do you use: reverse transcriptase.
We have to give it a primer. So remember, five prime to three
prime, we'd like to put a primer going over here.
Any ideas for a good primer? Poly(T), isn't that convenient?
One of the reasons that mammalian messages have poly(A) tails is so
that we are able to reverse transcribe them using poly(T)
primers. No, that's actually not true. So, we use reverse
transcriptase. And what we can do is we'll copy
this RNA into a strand of DNA. There we go.
Then what we'll do, next step, is we'll take the DNA,
and we'll copy back into a second strand of DNA.
And now, we have double-stranded DNA whose sequence matches the
already-processed mRNAs. Sorry? So, the sequences would
match the mRNAs. So what you could do is instead of
taking human DNA from the nucleus, you could take RNAs,
turn them back into DNA by reverse transcriptase,
and make a library now that consists of zillions of inserts,
each of which has what's called a cDNA, a copied DNA,
copied back from the RNA. The great advantage of this is that
the human cell has already done the splicing, and so there
are no introns left. Now, when you stick it in a
bacterium, the bacterium is able to express this. It's able,
if you give it its own bacterial promoter, to make an RNA.
And if you don't ask the bacteria to have to splice,
if you just give it a pre-spliced piece of DNA that doesn't need
splicing, it can translate that DNA. Now, notice we used all of our
tricks. You had to know about reverse transcriptase,
poly(A) tails, structures of genes, introns, exons, yes, question?
It doesn't. You do this in the test tube. You purify human mRNA in the
test tube. You take that mRNA in a test tube, add reverse transcriptase,
add poly(T), make this reaction of RNA to DNA in the test tube go back.
Where does it come from? Viruses that copy themselves back for a
living, right? So, again, every single thing we're
using comes from some living organism that does this
kind of stuff. And, when I teach you about the
facts of how viruses replicate or what the structure of mRNAs look
like or whatever, it's because every bit of knowledge
we get about the way biology works turns into an incredibly powerful
tool as it's turning out for us to actually be able to further study
biology. So, great. So, where does reverse
transcriptase come from now? Originally they come from viruses
that turn themselves back from RNA to DNA. Now, how do you get reverse
transcriptase? Catalog, right,
very good. All right, so this is called, finally,
a cDNA library. And, if you had made a cDNA library,
you would be able to screen the cDNA library to find the gene for insulin.
Is this useful? This happens to be,
for example, one of the consequences of this was the biotechnology
industry. OK, so if you have any doubts about
the usefulness of understanding these abstract things about E coli
and bacteria and stuff like that, one of the consequences was
Genentech, Biogen, and Amgen, and if you just simply
walk around Kendall Square, within a mile of this place you will
see laid out before you the consequences of this ability,
OK? It's transforming Cambridge. Yes?
And the world. Yeah. Indeed.
It might be that producing large amounts of insulin was bad for the
bacteria because there would be so much protein it would clump and kill
the bacteria. It might be that insulin, for various reasons,
might not fold appropriately in the bacterial environment.
And, this is why the biotechnology industry has lots of smart people
working in it because you're totally, 100% right. You might decide that
instead of cloning it in bacteria it's better to clone it in some
insect cell in culture which, in fact, people like to work with,
or some other cell, or a mammalian cell. And so,
I simplify by saying put it in coli, but in fact that might test six
different cell lines, six different host possibilities.
They might have to take the insulin out and refold it in vitro
and things like that. You're totally right.
This is actually something that requires work to do it right,
just like building an airplane requires work.
I could tell you Bernoulli's principles, but then Boeing does
more than just writes down Bernoulli's principles.
OK, so onward. Now, I'd like to turn next to analyzing your clone.
Analyzing the clone, so suppose we have, maybe it's by positional
cloning, maybe it's by cDNA cloning, but one way or the other we've got
us a clone that we're very interested in.
Maybe it has the insulin gene. Maybe it has the Huntington's
disease gene. Whatever it is, we're going to want to study it.
And at the moment, I haven't told you how I would even read its DNA
sequence or analyze its DNA. So, the first step is, of course,
I have to purify the plasmid. And, it turns out that that can be done.
There are simple biochemical techniques, as I mentioned in a
previous lecture, that allow you to grow up a lot of
the bacteria, crack them open, and the plasmid being a little
circle, and being a little more tightly super-coiled and wound up
has somewhat different physical properties. And you can use those
to purify the plasmid. So, plasmid preps are not hard to
do. You can get a fairly pure collection of the plasmid.
Now, suppose I've done this for, oh, I don't know, let's take my
first example, orange mutants. Suppose I tried to rescue bacteria
that were orange minus, and suppose I found that 50
different plasmids rescued my orange mutant because I transformed a lot
of plasmids in, I plated it, and 50 colonies grew up.
Are they all the same thing or are they different?
Is there any quickie way to take a look at these 50 plasmids and see if
they're identical or fairly close, or obviously different? Well, I'd
like to take some way to take the DNA from the plasmid and analyze it
kind of easily. I might want to see,
like, how big is the insert? Right, that'd be one way,
if they had different sized inserts so they couldn't be the same thing.
So, maybe what I could do is how do I clone this? I used EcoRI sites I
recall. So, I have EcoRI sites here. Suppose I were to take this DNA,
and I were to now cut the DNA from the plasmid with EcoRI.
Then, what I would get is two separate molecules.
I would get the vector and the insert. How could I see how big
they were? Gels, gel electrophoresis is the way to do
that. So, I take a gel. A gel is a slab of gelatin,
Jell-O, OK, and normally it's laid flat, but I'm going to do it
vertically here. I load into the top of it here a
little bit of my DNA, this whole mixture. I take the
plasmid. I cut it. I put it in here. DNA's positive
charge or negative charge? Negative. So, where should I put
the positive pull? On the bottom, well done.
That's often not done, and to the detriment of the experiment.
If you put the positive pull here, it goes the wrong way, and everybody
has to do that at least once. So, what'll happen is the DNA
fragments move through, and the smaller fragments move
faster than the big fragments, right? If something's little, it'll
move fast. If something's big, it moves slowly: little, big.
Smaller moves faster because it wiggles through the little pores in
the gel better. So, suppose I were to do this for a
bunch of plasmids, and what I saw was this.
First order, what do you guess? Sorry? Top road's probably the
plasmid vector. This is probably the vector,
and what do I know about the inserts? At least two inserts,
at least two distinct inserts. Now, if I wanted to be sure that was
the vector, maybe what I could do is take another row,
and run a known amount of the vector, take the vector alone and I could
check that the vector alone runs over here. And maybe I might take
some other known molecules. These would be called molecular
weight standards. So, if I run some knowns in one of
the lanes of the gel, I can even measure and say,
ah-ha, the insert is somewhere between the size of this one and the
size of that one. And so, I get a little ruler that I
can put on the gel. So, in fact, that's the first thing
you would do is you digest your clone that way.
Now, does the fact that these guys have exactly the same,
apparently, size on the gel mean that they're the exact same piece of
DNA? No, because you can't even actually tell it's exactly the same.
There's a limit to how precisely you can measure it.
So, what else could you do? You could try another restriction
enzyme. It turns out that since there are so many restriction
enzymes in the catalog, if I take a piece of DNA,
maybe that Eco fragment, I could try cutting it with HinDIII.
And when I cut it with HinDIII, I'm going to get three distinct
lengths. I could try cutting it with, oh, I don't know,
pick another enzyme, BamHI. When I cut it with BamHI,
I'll get some other lengths. And, how to get these lengths by
adding these, by running them out on a gel and looking at their sizes.
What if I added both HinDIII and BamHI to my test tube?
I'd cut at both sites. So, I'd cut here, here,
here, here, here. So, this is cut with HinDIII,
here cut with BamHI, here cut with both and I could measure these
lengths. So, suppose I gave you this as a computer problem,
I have a string and it's an unknown string, and I cut it at two places
and I get these lengths, X1, X2, X3. And then I take that same string and
I cut it at other positions, Y1, Y2, and Y3 are the lengths that
result. And then suppose I now cut it at both of the sites,
and I measure it, and I get Z1, Z2, Z3, Z4, Z5. If I gave you all
those numbers, could you figure out where the sites
must be? Probably. It turns out to be a reasonably
doable computer problem, although it can get a little hard in
places. And you could try a third enzyme and
a fourth enzyme, and it's a cute exercise to write
yourself a little piece of code that will figure out where the sites are
based on the lengths. The reason it occasionally gets
funny what if Z3 and Z4 are exactly the same length and they run on top
of each other in the gel, and there are special cases.
But you can kind of reconstruct where those restriction sites must
be just by writing a good piece of code that'll put these pieces
together. This is called restriction mapping,
and it's great fun. Everybody likes to do this once.
But, it's only a limited amount of information, right,
because you get where the sites are, and I guess if I gave you ten clones
and they all had exactly the same restriction maps,
the exact same positions of these restriction sites,
you'd feel pretty confident they were the same clone.
But you still wouldn't really know much about the clone other than it
had two HinDIII sites and two BamHI sites, and here's where they were.
What do you really want to know about this clone? It's
DNA sequence, right? Let's not settle for anything less
than the exact nucleotide sequence of the clone. So,
that's really the last key topic is sequencing DNA.
How are you going to sequence DNA? Well, suppose I give you some
double strand of DNA, five prime to three prime,
five prime, three prime, double stranded DNA.
Let me heat it up. What happens when I heat up DNA?
It melts the hydrogen bonds, the non-covalent hydrogen bonds here
break, and I got my two strands separated. Now,
what I'd like to do is I want to start reading out this DNA sequence.
So, I'm going to make me a primer. Now, golly, here's a primer.
You're going to ask me, how did I even know what primer to
use if I don't know the DNA sequence? How can I make a primer?
Hold that question. Make sure I remember to come back and answer
that, OK? But for the moment, grant me that I have a primer here.
What I'd like to do is add DNA polymerase. So,
let's add some DNA polymerase. And, I'd like to add nucleotide
triphosphates, dNTPs, the dATP,
dCTP, the dGTP, dTTP, and if I add DNA polymerase and I
add my nucleotides, what does Arthur Kornberg tell us
will happen? It'll start polymerizing,
right? And, it'll stop there. So, the polymerase knows the bases,
right? It knows what base to put in because polymerase is very smart.
So, the bases get put in correctly. The only problem is, how do we get
polymerase to tell us what it just did?
Here's a cute trick. This is, by the way,
a cute trick that won the Nobel Prize. So, suppose my primer is
like this: five prime, T, A, A, T, T, C, T, and the
template strand here, A, T, T, A, A, G, A, now let's keep
going, A, T, G, C, C, A, A, T, G,
G, A, T, T, A, five prime. So, there's my primer.
There's my template. I'm going to start adding. Well, let's
add our polymerase. Let's add our dNTPs,
polymerase, dATP, dCTP, dTTP, dGTP, and then I want to add a
special extra good old ingredient into this.
The special extra ingredient I want to add is a defective T,
a defective dTTP. What do I mean by defective? I mean chemically
modified in such a way that it can't be extended, that you
can't extend past it. So now, let's follow my reaction.
I'm going to start with, I'm just going to write them down here,
T, A, A, T, T, C, T. What's the next base I'm going to put in?
T, OK? Is that a defective T or a good T? I don't know.
It could be. Maybe it's a defective T, which I'll put a little star
there, OK? If so, what happens to my polymerase?
It stops. It can't go any further. It can't go any further because the
T's defective. But what if it wasn't a defective T?
What if it was a good T? Then what goes on? The polymerase
will put in, keep going guys. A, C, G, G, and what does it put in
now? T, right? Now,
is that a defective T? Maybe. We don't know.
If it is a defective T, it stops there. Otherwise,
polymerase goes here, and the next space is what?
T, and is that a defective T? Maybe.
And, if it's not a defective T, then polymerase goes on, puts in an
A, puts in a G, a C, C, and then a T.
And maybe that's defective. All right, which of these
possibilities is what polymerase does when I throw it in?
Well, all of them. There's a lot of molecules there.
Some of the molecules, by chance, happen to install a defective T,
and they grind to a halt here. Sometimes, a good T's put in and the
molecules stop here. Sometimes they stop here,
and if I start with a big collection of primers in a lot of my template
DNA, I'm going to get this whole collection of different molecules of
different lengths. What lengths do I get?
The lengths correspond precisely to the positions of the Ts.
I get a series of molecules whose lengths perfectly match the
positions of Ts. Well, first off,
how do I measure their lengths? Run a gel, bingo, run a gel.
So, if I could run a gel that could separate nucleotides based on length
that two next to each other, another one up there, I'd see a
small molecule, length one, two,
three, four, five, three, six, eight, I'd see one of
length eight. I'd see one of length, what's the next one,
13, eight, nine, ten, 13, 14, so eight, nine,
ten, 11, 12, 13, 14, 15, what's that, 13, 14, 15, 16,
17, 18. OK, those would be the positions at
which I would see this T. So, I'd need to have a special kind
of gel that's so accurate that it can separate single nucleotides,
right, that the lengths, but that can be done.
There's acrylamide, the polymer that will do that.
That'll tell me the exact lengths of the T's. What else do I do?
Well, let's obviously do it from the other bases.
Let's try defective A, defective C, defective G.
Let's see, if I got it right, which we'll try, it ought to end up
looking something like that. And if not, you get the picture,
that this ought to match up as to which columns have which lengths.
OK, I think I got it right. That tells me the lengths of the
molecules. So, I could read off at sequence.
The sequence of that molecule ought to be, starting over there,
the sequence of what I've added in, ought to be something like T, A, C,
G, G, T, T, A, C, C, T, yep, it worked.
It's exactly right. Bingo. I can now read the sequence.
Fred Sanger, a brilliant scientist, thought up this method of just
exploiting E coli's own polymerase or other organism's own polymerases.
So, copying and all the chemistry that had to be done was thinking up
a defective nucleotide that could not be extended.
It could obviously be inserted. It can't be extended. So, one
question is, what's a defective nucleotide? Well,
you will recall that our nucleotide in the sugar phosphate chain
is sitting like this. Let's see, hanging off the one prime
carbon is the base. This is the one prime carbon,
the two prime carbon, the three prime carbon, the four prime carbon,
the five prime carbon. What do we know in DNA at the two prime carbon?
Normally in ribose there would be a hydroxyl here,
right? But in deoxyribose, there's just a hydrogen.
So, if this is deoxyribose, so a dNTP really means a two prime
deoxyribose, where do I now attach my next base in the sugar phosphate
train? Three prime ends, and what do I attach it to: the OH.
What do you think would happen if there's no OH there?
You're stuck. All you've got to do is take off that hydroxyl.
No hydroxyl group. If you made nucleotides that don't have that
hydroxyl group, they can't be extended.
So, instead of these being just deoxy at the two prime position,
they are dideoxy, deoxy at two positions.
They are two prime, three prime, dideoxynucleotides.
That's it. Now, if you needed to get two prime three prime
dideoxynucleotides, they're in the catalogue of course,
right, because Fred Sanger had to make them himself and all that,
but you can just buy them now. And so, you can do the sequence.
A few other little details here, though, guys.
How do we see the DNA and the gel? One possibility would be staining
it. There are some dies like ethidium bromide,
and for doing your restriction mapping, using a dye that sticks to
DNA like ethidium bromide does is pretty good. And then you put it
under fluorescent light and you look. For sequencing,
the amount of DNA is so little that it's hard to see with a dye by the
naked eye, which is what you do with restriction map. So, sorry?
So, the first thing people did was radioactive. What they did was they
took a primer, made it radioactive,
and you did this whole sequencing reaction with radioactive primer.
Then, when you run the gel, you take your gel and you expose it for
some number of hours, eight hours maybe, a piece of x-ray
film, develop the x-ray film, and you'll see that picture. So,
one solution that you could do to visualize is using radioactive
nucleotides. So, we got the defective nucleotide.
We now need to visualize our DNA. Let's visualize the sequence.
One possibility: radioactive. The second possibility, someone
already mentioned it, a fluorescent dye.
Now, here, a fluorescent dye could be put on, and you can't read it
with your eye, but lasers are very good at reading.
So, you might run a whole gel here and have lasers scan it.
But, you can actually do better than that. Suppose I put my
fluorescent dye on my dideoxynucleotides.
Suppose I put it on my dideoxynucleotides,
and suppose I even had enough chemistry at my disposal that I
could put a different color of fluorescent dye on each
of my nucleotides. Then, whenever the dideoxy is put in
to terminate the chain, it carries with it its own color.
Wouldn't that be cool? And, that's what's done. Not just can you buy
dideoxynucleotides now, but you can buy the four different
dideoxynucleotides each with its own dye attached to it.
So, there are di-dideoxies I guess, sorry, but it's different di's,
right? They're dye-dideoxies. So, you could do that.
And then what you get would be that in this column you get this color.
And in this column, you'd get this color. And in this column you'd get
this color, etc. I'm not worrying about where they
are here. And they'd all be different colors and it would be
very pretty. You know what? Why do we need to run separate
lanes anymore? If we got a laser,
we can tell the laser scan it to tell it different. Stick
it in one way. In fact, what's done is stick it in
a capillary tube, throw in all four at the same time
now, and as these fragments come by, each has its own color. And all we
need is a laser scanner capable of sitting right here. Here's
my laser scanner. And the laser scanner,
positive here, negative here, as the DNA flows by through this
polymer, the laser scanner reads off which colors just went by.
And it goes A color, C color, T color, G color. That's it. So,
there are actually machines now that have 96 different capillaries.
These are called capillary tubes. And you can have 96 of them with
laser scanning across, and in each column now,
it turns out that you can read almost 1,000 letters,
1,000 bases per column per capillary times about 100 capillaries.
Or in other words, you can read out about 10^5 bases of information.
You can read out 10^5 bases of information in about two hours.
Of course, you can do that ten times a day. So,
you can actually read out 10^6 or about a million bases of information
per machine. And here at MIT, we have 100 of these machines. So,
we actually can read out a little shy of 100 million letters of DNA
sequence per day, which I mean is a lot.
We read about 40 billion letters per year here at MIT,
and this is how we do it. How much does a machine cost?
List, or do you want a deal? They list for $300,
00, but if you buy in bulk, I can do better. [LAUGHTER] We buy
it in bulk, by the way. So now, how are we going to get our
primer there? That was the only little bit we were missing is where
did our primer come from? The last little detail: here's my
vector, remember, and I want to sequence this insert.
How am I going to get a primer in the insert? I don't know what its
sequence is. How do I even start this? Sorry? Well,
but that won't tell me what the sequence is that I have to,
I mean, I was looking to try to get a primer that matches the insert.
And I don't know what the insert is. So, how am I going to get a primer?
Oh, I know the vector. The vector is well known.
It sequence is in the catalog. Let me instead just use a primer
that happens to sit in the vector, and I'll match to a known sequence
to start with, and then I'll sequence into my
unknown territory. So, this is how you get the initial
primer was you arrange that your initial primer is sitting in known
vector sequence. All right, so you can now sequence
DNA. I've got to say, I've taught this course for a little
more than a decade, and being able to say,
now we can routinely sequence about a million letters per machine,
and 100 million letters per day, and things like this was not
routinely the case. When we started teaching this
course, I was describing what we di