| Michael W. Hunkapiller |
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[figure 1]
[figure 2]
[figure 3]
[figure 4]
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Thank you for
that kind introduction, Dr. Matsubara. I would also like to extend
my thanks to Dr. Takeda and the Directors and staff of the Takeda
Foundation for putting on this wonderful symposium and inviting us
to Tokyo. [figure 1] To give you a sense of the passion that Craig
(Venter) and I have developed over the last several years for the
life sciences research field, I will tell you that we think the benefits
to mankind that can come from leading edge research to understand
fundamental human biology, carried out literally around the world,
are enormous. From this understanding, we will be able to develop
the kind of insights and tools that will allow people to have improved
lives and longevity.
I will begin my comments by saying that when Craig (Venter) was first
presented with the technology and the idea of sequencing the human
genome, he was shocked. However, we looked at the possibility together
and decided maybe it was doable. We may have been crazy at the time,
but with Craig's hard work and the team he was able to put together,
sequencing the human genome was, in fact, a doable project.
In my remarks, I am going to try to give you not just a sense of how
the development of the technology to carry out the sequencing of the
human genome occurred and what was involved, but also a sense of where
people will go from here. I will present the future development of
the technology in terms of what is needed to help further the understanding
of this blueprint of life, i.e. the genetic code.
The goal of the researcher -- to understand how biological systems
work at the fundamental biochemical level -- is one that Applied Biosystems
supports with its technology. [figure 2] In order to achieve this
level of understanding, researchers need to know the structure and
the function of several key, complex, biological molecules. The most
important of these, from an information/content perspective, are nucleic
acids, more commonly referred to as DNA and RNA. Each has a separate
job. DNA's role is to act as a fixed repository of information that
can be transferred from one generation to another. RNA's task is to
carry out the DNA's instructions for particular biological processes
at given stages in a cell's life. As Craig has discovered, and as
the Human Genome Project has discovered, there are only about 30,000
different genes that are the core building blocks of DNA structure.
These genes give rise to the transmittance and the use of the genetically
stored information.
The information that's housed in nucleic acids is translated into
activity when a cell wants to put it to use in the form of a different
kind of complex, biological molecule called a "protein." Proteins
are then sometimes broken down into smaller groups of peptides and
can be further modified with the addition of other kinds of chemical
groups to carry out the specific function for which the protein is
intended. While there are only about 30,000 genes in the human genome,
there are upwards of a million or so different proteins, peptides,
and modified forms of these compounds that can be active in a cell
at one time or another during the course of an organism's life.
Proteins function not only as individual molecules to carry out a
specific chemical reaction or perform a specific structural task,
but they also form networks in which they work together in different
ways, at different times, under different conditions in order to carry
out much more complicated tasks. Therefore, unraveling biological
function requires a lot of information, both about the specific structural
components of the individual molecules that make up these nucleic
acids, or protein networks, and about the three-dimensional structures
that give rise to a lot of the functional characteristics that are
imparted by that structure contained within them.
When we looked at the problem of how one looked at these processes
from a genome-wide or proteome-wide, or cell-wide level, several years
ago at Caltech, it was pretty clear that biology, at that time, was
a manually driven process. Automation had made inroads into the drive
to improve chemistry, but hadn't hit biology as much as we thought
would be necessary in order to carry out large-scale studies. Automation
was needed to begin to get a look at not just the very tiny pieces
of living cells (e.g., one protein, one gene, or one cell), but to
study the entire component of those molecules. [figure 3] So, while
at Caltech, Lee Hood and myself, and others, proposed the concept
of transforming the way biology is done at the basic research level
to take advantage of modern tools for doing large-scale structural
and functional analysis of these molecules.
I would like to give you some insight into three classes of these
tools, with a good part of the focus illustrated in this slide, and
to talk about the tools necessary for doing structural characterization
of genes and related nucleic acid fragments. [figure 4] The basic
tool, as was mentioned by Dr. Matsubara, is one known as "DNA electrophoresis,"
which separates molecules on the basis of their size. [figure 5] It
was the fundamental understanding of the structure of DNA, by Francis
Crick and Jim Watson in the early 1950's, that laid the groundwork
for this kind of tool to be widely used in the study of nucleic acids.
Contained within nucleic acids, in the order of the components that
make up one gene versus another and one part of the chromosome versus
another, is the simple, but elegant, ability to carry the information
necessary for their own replication. It was this realization and the
development of tools by Fred Sanger, at Cambridge, and others in the
mid-1970's that gave rise to the use of DNA electrophoresis as a tool
for studying the basic ordering of the components within nucleic acids.
They each have only four constituents that make them up. It's the
order and the number of these constituents that give rise to the differences
between one gene and another.
The concept, here, was to use a lot of the fundamental biochemistry
that's involved in normal cell replication of nucleic acids as a means
of identifying and elucidating the structure of these molecules in
terms of their basic sequence. [figure 6] What was generated, and
what's called the "Sanger chain terminating sequencing methodology,"
is a series of fragments of DNA, with the original implementation
carried out with four separate reactions, one for each of the four
types of nucleic acids that can make up DNA. By separating the fragments
by size using electrophoresis, and realizing that the smaller pieces
have an order relative to the larger ones, and that this order is
related to the sequence, you can determine the sequence of the DNA.
The shortest piece would be an A, the next piece might be a C, the
next one an A, a T, and so forth. By manually going through and reading
the ladder from top to bottom you could determine the sequence. The
beauty of this technique was that it worked; the drawback was that
it required somebody to sit there, look at this picture and the separated
ladders of DNA, and manually interpret the sequence. You can see,
without much knowledge of the details, that the signals seen in the
form of these bands are not uniform. If there are any anomalies, in
terms of how fast the bands run in part of the gel versus another,
it might make the sequence complicated to read. In fact, this was
often the case. Thus, the Sanger method was a fairly error-prone process
in the beginning. |
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