I'm Ron Vale, and today I have the pleasure to be speaking
with Dr. Wally Gilbert. Dr. Gilbert started off as a
theoretical physicist, and then through an introduction
from Jim Watson, moved into the area of molecular
biology, where he's made many important discoveries
over the years. He isolated a protein that can control
a genetic element, the lac repressor. He's also
made many important contributions in our understanding
of mRNA. But today we're going to be talking about
his seminal work on DNA sequencing. Work that
rapidly went to his being awarded a Nobel Prize
in 1980. So thank you for joining us today, and
maybe we can just start off by going through the history
of what led you into tackle the problem of DNA sequencing.
Okay, in the early 70s, we knew of course that the
sequence of the DNA carried the ultimate code that's passed
from parent to offspring. But in fact, because it's essentially impossible
to actually work out the DNA sequence. I had isolated
the lac repressor, the protein that binds to a region of DNA.
In the early 70s, I actually then tried to isolate that region
of DNA to which the protein bound, and we did that by
attaching the protein to DNA, dissolving all of the rest of
the DNA with DNase and then isolating the little fragment
left, protected by the protein. So I had that physical fragment in my hand.
And I wanted to know its sequence. We tried developing
DNA sequencing methods. Nothing seemed to work.
And we then worked out its sequence, my technician,
Allan Maxam and I, for about two years. By copying that little fragment
of DNA to RNA, and then working out the sequence of
the RNA by breaking it with enzymes and using gigantic
electrophoretic apparatuses to separate the RNA spots on
paper electrophoresis. It took us two years to work out
essentially 24 bases of DNA. That's about one base
a month. We knew that a typical gene in bacteria, we thought,
the genes in our body would be in the order of 1000
to 15,000 bases long. We now know of course, the genes in our body are
30,000 bases long, far bigger than anyone could've imagined at the time.
But at one base a month, you would never get to a thousand
bases of DNA. You would never be able to sequence that in any way.
And that was actually the nature of the problem.
In the early 70s. Another group worked out the sequence
across the entire beta-galactose genes. Again by copying
DNA and RNA. And I thought, well, that's all I interested in,
the way it sequences. Go on and work on something else.
A Russian, Andrei Mirzabekov, came to visit me. And he wanted
to convince me to do an experiment. He wanted to convince me
to look at whether the repressor bound to DNA in the large
groove or the small groove by modifying the DNA with chemical
agents. And the agent he liked was dimethyl sulfate,
which puts a methyl group on the guanine in the large groove.
I thought it would be an awfully interesting experiment, but I
didn't see a way of doing it, so I didn't do anything.
Six months later, the part of his trip to America, came through
again. We had lunch, he tried again to convince me I should
really do an experiment of that kind. I had the repressor,
I had the DNA, I had all access to all of this. The question
-- the problem was, how could I recognize where these
modified bases were in the DNA, which the repressor would bind.
In the conversations with Andrei, we thought of a way of
actually trying to identify the positions of these modified bases.
We had available ways of breaking the DNA with restriction enzymes,
such that we could isolate a piece of DNA about a hundred
nucleotides long. Which could be labeled at one end or the other
with radioactive phosphate, depending on the way in which we
isolated the DNA and broken it sequentially with restriction enzymes.
So we could have available a DNA molecule we knew would bind
the repressor with a label at one end, and the modification
with dimethyl sulfate we knew would weaken the base,
which the modification occurred in such a way that we could
break the DNA at that point. So we reasoned since we knew
the whole sequence of this tiny little molecule, we should be able
to break the DNA at the point at which the methylations
occurred, and separate those molecules by size on a gel.
And since we knew the sequence, we should be able to
decipher which molecule came from what. So I did that
experiment. I put the repressor on the DNA, modified it,
second tube had no repressor on the DNA, modified it
with dimethyl sulfate, broke the DNA, ran gels, and the experiment
was a great success second time I did it. First time it didn't work
at all. Second time I did it, it worked perfectly. The guanine
modified the large groove, the repressor blocks the modification of
guanine. The adenines were modified in the small groove,
you can see yes, the repressor blocks certain adenines.
But the striking thing about that pattern, which I had expected
to have to deduce from the sequence what the pattern should
be, the striking thing about the pattern on the gel was
that it's separating the DNA fragments from the smallest
to the largest, you've got a very characteristic pattern.
And you could see, oh here's DNA broken at position 30.
Here's the next band, just a little higher, broken at position 31.
Well, you look at that pattern and you say, this must
be the sequencing method. It's sufficiently clear that
in one go I could read essentially a hundred bases of DNA.
So was there a eureka moment?
Well, there's a moment when you look at that and you say,
clearly this is a sequencing method. The only problem is
can you actually sequence just by looking at the A's and
G's? We actually sequenced something which we never
published just by looking at the A's and G's coming down
the two strands. And we started looking for chemical reagents that
would break the DNA at cytosines and thymines. Now, in fact,
we knew of such reagents immediately, because I knew hydrazine
broke DNA at cytosines and thymines. That's part of the
old chemistry that we tried five years earlier, trying to work out
a sequencing method. So we could immediately get a display
of the C's and the T's. Allan Maxam then spent about 9 months
trying to find a way of changing that display so you could
distinguish the C's and the T's. Essentially by throwing
every chemical in the laboratory into the reaction to see
if it made a difference. And he came up with a modification
by adding salt to the reaction. And we ended up with effectively
four reactions, each one of which was specific enough
to reveal a breakage of DNA at a certain base. And
the trick behind the sequencing was this idea of measuring
a distance from the wavelength of the labeled phosphate
to the position of each base. The first gels would display
molecules on the order from 10 to 50-60 long, you could read that
sequence. We very quickly went to gels that could display molecules
hundreds long. And you could read in an afternoon
more than one base a month, and in an afternoon
you could read several hundred bases. The next
I had had a previous student, Nancy Maizels, who had worked out
63 bases of DNA by copying an RNA for her thesis.
The next thesis was about 1500 bases of DNA, was
the sequence of the repressor gene itself worked out from the
DNA. In that case, correcting the protein sequence, which
there were mistakes. And that was the next student's thesis.
The student after that did 5000 bases of DNA that did an entire
plasmid. We stopped giving PhDs for sequencing. The method was actually
developed around 1976, '75 or '76, and we gave the method
away at Gordon Conferences. The Gordon Conference that
summer. We gave a little handout and people began to sequence
DNA immediately. The paper, the official paper published on it
was probably not published until 1976, or 1978. By that time,
people came to the laboratory to learn the sequencing
methods and took it away.
How long did it take from the time when you were working
on the problem from the repressor point of view to developing
this general method?
The total time was 9 months, to find the last bit of the
chemistry. And the sequencing came -- the fact that you could
do the sequencing came immediately out of that first experiment.
That immediately said, oh it's clear that the patterns are distinct
enough. Fred Sanger was working on the problem
simultaneously, trying to develop sequencing methods based
again on ultimately the same idea, can I measure a distance
along the DNA to a position at which a base occurs?
The gels that he used came out of my laboratory, gels that he
also used came out of the technology that we had developed
in terms of sizing DNA molecules. And most of that technology
had been developed earlier just to size DNA molecules
for the whole problem of finally isolating the DNA fragment
that was bound to the repressor.
And what was the reception from the community when
you first showed these gels at meetings?
People began to sequence immediately. I mean the sequencing
took off at that point, both Fred's method became available
and our method at essentially the same time in 1976.
And people around the world began to sequence actively
DNA. The rate of DNA sequencing started to go up following
Moore's Law, doubling every 18 months. So from that time
until probably 2010, it may be going even faster now.
So just looking at how the field has progressed since then,
when you first developed this method, did you have any
idea or anticipation of how the field of DNA sequencing
might develop?
It's a hard question. In the middle '70s, when we developed the method,
and by the late '70s, it was clear you could decipher
any gene. And that's what people were doing all over, everywhere.
And the Nobel Prize is actually for the recombinant technology
It was given half to Paul Berg for standing in for all the
people who showed you could move the DNA fragment
around from one cell to another, put it in a plasmid,
grow a human DNA fragment in a bacterial cell.
And to Fred and myself for the ability to decipher the DNA.
It wouldn't do much good if you could move DNA around
if you couldn't decipher it. Across that period people were
then sequencing genes like mad. Around 1980, it was essentially
the workhorse, the sequencing methods of those times. Fred's
and mine were being used equally. And continued to be
equally until the middle '80s, when the machines came in and
which the dideoxy method was mostly adapted to.
And once the machines came in, Fred's method took over.
Almost entirely, the chemical method is used only for very special
cases. It could do certain things that the dideoxy method
can't do. It can reveal the methylation pattern and things like that.
But beyond that, it became sort of for specialty use.
I did not anticipate at the time we discovered the DNA
sequencing, the genome. In fact in 1985, I was invited
to a meeting in California by Bob Sinsheimer, and
with a sort of idea, could one sequence the human genome?
And I went to that meeting thinking, that's the silliest
thing I ever heard of. That's so much larger than things we're
sequencing up till now. And at the meeting, I thought about
where the technology has gotten to. I thought finally,
it's probably possible to sequence the genome. I became,
at that time in 1985, an advocate of the idea that yes, you
should sequence the genome. Originally, I thought it would be
a 30 year problem, as I suggested to Bob. And he said
to sequence the genome, 10 years for the first 1%,
10 years for the next 10%, and 10 years for the last. And do it
on an industrial scale, but a 30 year problem.
It actually took about 15 years, it was a purely industrial
scale problem. And I realized in '85, the technology that
then existed was improving at a rate such that you could do
the entire human genome in a reasonable amount of time.
Of course by now, the technology has changed radically from
the first ones we had. We're now at least at third or fourth
generation of sequencing, the machines and technologies
out there. And whereas, originally it was one base a month,
it's now billions and billions of bases in an afternoon.
I find it sort of unimaginable to have been part of this entire
effort, but in fact, to have seen it go from nothing. At the time,
we were doing this first DNA sequence, Jim would be
only too willing to assure me that people would never be able,
off in a decade, be able to sequence genes.
It gives an amazing example of how fast science is moving
these days.
That's right.
Especially in the biological sciences.