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IVD Technology Magazine
IVDT Article Index
Originally published September, 1998
DNA-chip technologies
Part 1: Research fundamentals and industry
catalysts
Cliff Henke
Is the adoption of DNA-chip technologies a nano-pipe dream, or the
next medical revolution? In this three-part series, IVD Technology
explores the potential of this emerging technology for diagnostics.
Part 2 is also available for on-line viewing.
As is often the case with breakthrough technologies, a great deal of
hyperbole has surrounded the development of micro- and nanoarray
diagnostic technologies, popularly known as DNA or genetic chips. Yet
much of the optimism expressed in such statements is almost certainly
justified. Industry analysts agree that when their promise begins to be
fulfilledprobably sometime early in the next decade DNA chips will
usher in a new era in medical care.
Researchers in the field expect that DNA chips will enable
clinicians--and in some cases even patients themselves--so quickly and
inexpensively detect the presence of a whole array of genetically based
diseases and conditions, including AIDS, Alzheimer's disease, cystic
fibrosis, and some forms of cancer. Moreover, the technology could
make it possible to conduct widespread disease screening
cost-effectively, and to monitor the effectiveness of patient therapies
more effectively. In the meantime, however, considerable work remains
to be done. So far, only a few companies have commercialized
DNA-chip products, and the barriers to market entry remain great.
This article, the first of a three-part series on DNA-chip technologies, will
review the theoretical underpinnings of the technologies and examine the
market forces that are driving product development. The second
installment will look at the state of the art, including the various
competing technologies in this embryonic field. The final article will
examine the obstacles to commercialization that companies in this new
marketplace will have to overcome, as well as prospective near- and
long-term applications for DNA-chip technologies.
Although the DNA-chip marketplace is in its infancy, with considerable
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challenges remaining to be overcome, the speed with which
manufacturers are progressing toward commercialization will soon make
DNA chips viable alternatives to traditional chemical assays. Indeed, if
analysts are correct, within a decade they will usher in a new era in
diagnosis and treatment for diseases and conditions that have genetic
origins.
Theoretical Principles
A variety of recent technological breakthroughs have made possible the
development of DNA chips. Fundamentally, however, genetic chips are
the result of achievements in two fields: molecular biology and
microfabrication technology.
Molecular Biology. Especially as it has been catalyzed by the work of
the Human Genome Project (HGP), research in molecular biology has
laid the groundwork for the development of clinical laboratory tests and
therapies involving genetic probes. Fundamental advances include the
use of polymerase chain reaction (PCR) or other amplification
techniques to make copies of a nucleic acid sample, which can then be
tested using a genetic probe, that is, a known gene and its molecular
structure. Also essential to the development of DNA chips has been the
creation of gene sequencers, machines that have automated the
biochemical tests necessary to identify genetic sequences using gene
probes.
But the roots of gene probe technologies go back much further, to
molecular biology discoveries made decades ago. Most important of
these are the base-pairing rules discovered in the 1950s by James Watson
and Francis Crick. Watson and Crick determined that the DNA
molecules found in living organisms are composed of a structure of two
twisted strands (the famous double helix) latticed together with pairs of
nitrogenous bases: adenine, cytosine, guanine, and thymine. They also
discovered that these bases always recur in the same two pairs: adenine
with thymine, and cytosine with guanine. Thus, by knowing part of the
molecular structure of a specific genetic segment, one can determine the
other part.
Moreover, these uniquely complementary strands of DNA can be sought
out by using one of the strands to test for its biochemical mate; this is the
basis of a gene probe. The process of one strand of DNA matching up
with its counterpart strand is called hybridization. It is this technique that
is used to determine a base-pair sequence in a DNA sample, also called
genetic sequencing.
Hybridization can be performed either in solution or on a solid support.
In traditional gene sequencing, the most common format for
hybridization is the Southern blot, which uses a nitrocellulose sheet.
However, some companies use solution-based processes, and there is
considerable experimentation in the field to develop new hybridization
formats. DNA-chip manufacturers are also exploring variations of both
solid-phase and solution-based hybridization for use in their
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microassays. At present, however, most DNA-chip companies use a
solid-phase technique.
DNA chips are designed to identify hybridization products in the same
fashion as with traditional sequencers. Once hybridization has been
completed, phosphorescent chemicals that bind to the hybridized
sequences are scanned with a light source, making it easy to detect their
presence with automated colorimetric or fluorimetric equipment.
Microfabrication Technologies. The second technological trend that is
making DNA-chip products possible encompasses the steady
improvements in nano- and microscale fabrication techniques.
Developed initially for use in computer chip manufacturing, these
techniques are now being exploited in a variety of other disciplines,
including DNA-chip manufacturing. These achievements have made
possible the application of organic structures (e.g., segments of DNA and
reagents) onto a substrate of inorganic materials. Unlike computer chips,
which use silicon-based wafers, DNA microassays are fabricated onto
glass or plastic wafers or are placed in tiny glass tubes and reservoirs.
Although the fundamental principles of molecular biology apply to the
design of all DNA chips currently under development or commercially
available, approaches to the fabrication of substrates for such products
vary considerably. While some developers use manufacturing techniques
very similar to those used in computer chip fabrication, others are
exploring techniques very different from semiconductor manufacturing.
At the computer chip end of that continuum is the approach taken by
such companies as Affymetrix (Santa Clara, CA). To produce its
Genechip line of products, Affymetrix bonds hundreds of genetic
sequences onto the surface of a microchip using photolithographic
processes such as photosensitive masks, chemical doping layers, and
other techniques used in computer chip fabrication.
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How Genetic Sequencing Works
Sequencing, the process of finding the molecular structure of a
DNA fragment, employs the Watson-Crick rules of hybridization,
whereby each strand of DNA can bond only to a chemical mirror
image via two sets of four bases: adenine (A), cytosine (C), guanine
(G), and thymine (T).
Step 1: Determine chemical structure of
fragment.
Representing all or part of a DNA strand of
interest, short fragments of DNA (typically
involving 525 base pairs) are identified.
Step 2: Separate strands.
DNA is denatured (separated) and
placed in solution or on a solid
substrate, forming a reference
segment for the DNA fragment of
interest.
Step 3: Introduce sample.
Unknown DNA sample is introduced to the
reference segment. If present, the complement of
the reference segment will hybridize (bond) to it.
Step 4: Identify result.
Chemicals that bond to successful
hybridization help researchers identify
results. Such chemicals are typically
photosensitive (fluorescent or
chemiluminescent), which helps
researchers confirm results.
Probe arrays are manufactured by Affymetrix's proprietary,
light-directed chemical synthesis process, which combines solid-phase
chemical synthesis with photolithographic fabrication techniques
employed in the semiconductor industry. Using a series of
photolithographic masks to define chip exposure sites, followed by
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specific chemical synthesis steps, the process constructs high-density
arrays of oligonucleotides, with each probe occupying a predefined
position in the array. Multiple probe arrays are synthesized
simultaneously on a large glass wafer. This parallel process enhances
reproducibility and helps achieve economies of scale. The wafers are
then diced, and individual probe arrays are packaged in injection-molded
plastic cartridges, which protect them from the environment and serve as
chambers for hybridization.
Another manufacturing approach involves the deposition of gene probes
onto the chip substrate using a tiny droplet sprayer that resembles an
ink-jet printer. This approach is being used by Combion (Redwood City,
CA), Rosetta (Seattle), ProtoGene Laboratories (Palo Alto, CA), and
Affymetrix. (Two of these firms illustrate the heavy involvement of
higher education in this emerging field; Combion was created from
research conducted at the California Institute of Technology, while
Rosetta uses techniques developed at the University of Washington.)
Manufacturers spray a chemical solution containing the gene probes in a
pattern onto the chip substrate, in the same fashion as in other clinical
lab tests.
Some companies, such as Nanogen (San Diego), use robots to deposit
the gene probes onto the substrate. Nanogen uses electrophoresis to
speed up hybridization.
Yet another approach is the use of gels in a solution-based process.
Scientists at the Argonne National Laboratory (Argonne, IL) hope to
find commercial backing for this approach within the next two years.
Thus, the concept behind DNA chips is simply that of miniaturizing the
gene sequencing technologies already being developed, so that many
assays and their related procedures can be performed together. DNA
chips will give researchers the ability to analyze thousands of genes at
once, and may also make it possible to conduct very elaborate diagnostic
procedures in such small settings as a physician's office or even with
mobile equipment used at the point of care.
Product Development Catalysts
Enormous forces are impelling the development of DNA chips. The
most important of these is the federal government's Human Genome
Project. Begun in 1990, the HGP is a federally funded and directed
research endeavor involving scientists throughout the world. Its goal is to
locate and characterize every gene on every human chromosome by
2003. Thanks to technologies such as DNA chips, researchers are now
hinting that the project may be completed by the end of this decade,
three years early.
The HGP has contributed in two significant ways to the development of
microassays. First, it has created a genomics market. Genomics is the
science of discovering, locating, and characterizing genes in organisms.
HGP grants issued by the National Institutes of Health and other
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institutions all over the world have increased the appetite for genetic
chips as part of the HGP's race to completion. As basic research dollars
of the HGP have led to more information about the human genome,
pharmaceutical companies and venture capital funds have poured in
further billions of dollars to commercialize applications of genomic
discoveries.
How DNA Chips Are Made
Step 1: Make gene probes.
Using conventional techniques such as polymerase chain reaction
and biochemical synthesis, strands of identified DNA are made and
purified. A variety of probes are available from commercial sources,
many of which also offer custom production services.
Step 2: Manufacture
substrate wafer.
Companies use
photolithography and
other nanomanufacturing
techniques to turn glass
and plastic wafers into
receptacles for the DNA
probes.
Step 3: Deposit genetic
sequences.
Manufacturers use a
variety of processes
ranging from
electrophoretic bonding
to robotic deposition to
adhere genetic material to
the substrate. Cleanroom
conditions and standards must be observed to attain the degree of
contamination control needed during the deposition process.
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Step 4: Customer use.
Completed chips are
checked for quality,
packaged, labeled, and
sent to clients. In use,
chips enable researchers
to identify the
components of probes
deposited on the chips,
usually with the help of phosphorescent tags.
In turn, HGP discoveries have helped researchers develop more and
better tools for their gene-hunting work--the second manner in which the
project has contributed to the development of DNA chips. Several
companies have either designed and manufactured chips to help tackle
specific questions for research establishments engaged in the HGP, or
have done so to help pharmaceutical and diagnostics manufacturers in
their commercial research projects. Moreover, the additional information
acquired in both government-funded and private-sector research has led
to product ideas with commercial potential outside medicine.
Toxicologists, for instance, have sought to adapt DNA-chip technologies
for use in agricultural research and in conducting environmental impact
studies.
For some companies, the key by-product of HGP research is directly
related to the field of medical diagnostics. Nanogen, for example, will
use its platform technology created initially for genomics research as a
base for developing commercial applications in the infectious disease
diagnostic market. According to Kieran Gallahue, executive vice
president of Nanogen, the company is currently looking into partnering
with some other large diagnostic companies. However, he adds, the
company will not be ready to market any products until late 1999.
Another large group of contributors to DNA-chip development has been
made up of pharmaceutical companies. Most microarray manufacturers
already have partnerships and long-term commercial arrangements with
the world's major drug companies to develop products that meet
pharmaceutical research needs. For example, several such teams are
working on ways to more quickly identify therapies that are effective in
identifying and preventing cancer-causing mutations in the p53 gene.
Other government programs have also helped develop genetic chips. For
example, Affymetrix and Hyseq (Sunnyvale, CA) each received $2
million in funding from the Department of Commerce's Advanced
Technology Project, which awards matching grants to firms whose
promising research is not sufficiently developed to attract commercial or
investor support but might show great economic promise in the long run.
The Department of Energy's national laboratories have been another
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catalyst of research and development in this field. Argonne National
Laboratory's work, mentioned earlier, is one example of this. The Sandia
National Laboratories have also helped some companies with research in
microlithography processes that can be used in DNA-chip fabrication.
Future Challenges
As DNA-chip companies prepare to bring their products to market,
major technological and regulatory challenges still lie ahead of them. The
technology trade-offs will involve finding ways to increase the number
of arrays on a single chip, as well as increasing the rate of production to
meet expected demand. Currently, few manufacturers are producing
chips beyond a pilot-scale production rate.
A second technology challenge involves achieving all these
market-oriented parameters at a cost that supports a commercially
acceptable price. Currently, DNA chips cost between $100 and $450
each. In a tight managed-care marketplace that places a premium on
technologies that can either show quick savings or more-efficient results,
some analysts say that such unit prices will limit the growth of the
DNA-chip market.
As products are readied for the marketplace, they will also encounter
regulatory challenges, such as ensuring that manufacturing processes
meet current quality systems and CLIA standards. These regulatory and
technical manufacturing hurdles will be discussed further in subsequent
articles scheduled in this series.
Conclusion
Although the DNA chips now commercially available are less than a few
years old, there is already a growing commercial interest in these devices
for research, diagnostic, and therapeutic applications. Enormous hurdles
must still be overcome, as will be discussed in the next installment of this
series. However, the pace of activity is so great that observers are already
confident that widespread application of DNA chips is less than a decade
away.
Bibliography
Cookson C, "Markers on the Road to Avoiding Illness," Financial
Times, March 31, 1998.
Douglas MG, "The Human Genome Project: Gateway to Managed
Health," IVD Tech, 3(4):4651, 1997.
Stipp D, "Gene Chip Breakthrough," Fortune, March 31, 1997.
Strategic Assessment of the Developing DNA Microchip Market,
Mountain View, CA, Frost and Sullivan, 1997.
Tortora G, Funke B, and Case C, Microbiology: An Introduction, Menlo
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Park, CA, Benjamin/Cummings, 1986.
Cliff Henke is a freelance writer based in Southern California.
Illustrations by Robert Margulies.
Continue to Part 2 of this series.
Copyright 1998 IVD Technology Magazine
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