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Many human diseases stem from mutations in an individual's DNA. These diseases may be inherited diseases such as cystic fibrosis, diabetes, and muscular dystrophy, or acquired diseases such as cancer and AIDS. Researchers in both academic and commercial settings are identifying the genetic basis of increasing numbers of human diseases at a dizzying pace--a trend that has yielded the recent discovery of the genes linked to adult leukemia, Huntington's disease, colon cancer, and Lou Gehrig's disease. Such strides in genetic research call for tools that can help scientists work more efficiently in their quest to understand the relationship between certain genes and human disease. One such tool is a new technology that enables researchers to analyze human genes using a microchip, and it's now on the way to the marketplace.
Microchips developed by a team at Affymetrix in Santa Clara, California, may some day replace traditional blood tests or other laboratory diagnostic procedures. Affymetrix's GeneChip products enable detection and characterization of large amounts of genetic information, according to Richard Rava, Affymetrix's vice president for research and engineering. They provide a dramatic departure from traditional, more cumbersome laboratory analysis methods used to extract genetic information. The GeneChip products are developed using a combination of photolithographic techniques common in the semiconductor industry and chemical synthesis: huge numbers of oligonucleotides, or DNA molecules, are synthesized very rapidly on the surface of a tiny microchip approximately one centimeter square.
The technology relies on "combinatorial chemistry, or the synthesis of many different chemicals in a minimum number of chemical steps," explains Rava, a physical chemist with extensive experience in instrumentation systems and software engineering. "Tens of thousands of different oligonucleotides are synthesized in a very small area."
This innovation is based upon Affymetrix's very large scale immobilized polymer synthesis (VLSIPS) technology. Introduced in 1991 by Affymetrix Scientific Director Stephen Fodor and colleagues at Affymax, parent company to Affymetrix, VLSIPS allows for the rapid and automated synthesis of large arrays of chemical entities on the surface of a tiny microchip.
Transforming Genetic Research
Although the Affymetrix chip technology was first applied to discovering new drug therapies, the company recently has worked to tailor the chip for use in medical research and clinical diagnostic settings. Researchers and laboratory technicians will use the GeneChip system as a standard format for analyzing large amounts of genetic information in a single test, boosting cost effectiveness, speed, and reliability.
According to Paul Berg, professor of biochemistry and director of Stanford University Medical Center's Beckman Center, Affymetrix has just begun to explore the utility of this technology in the research market. "The commercial market will require a different capability," says Berg, who received the 1980 Nobel Prize in Chemistry and serves on Affymetrix's board of scientific advisors.
"It's not going to be that inexpensive," Berg says of the technology. "We'll maybe have 25 to 35 machines going to research markets [at first]," he explains. "Ultimately, as the price goes down, one would expect that lots of hospitals could have one in their laboratory."
Affymetrix and partner Molecular Dynamics Inc. will be joined by researchers at the California Institute of Technology, the Lawrence Livermore National Laboratory, Stanford University, the University of California at Berkeley, and the University of Washington to develop the next generation of DNA diagnostic devices.
This team recently received a $31.5 million award under the National Institute of Standards and Technology's (NIST) Advanced Technology Program, which is administered by the U.S. Department of Commerce Advanced Technology Program. Members of the consortium will match that sum dollar for dollar.
Stan Abramowitz, program manager of NIST's Advanced Technology Program, explains why the Affymetrix-led consortium won one of the program's 13 grants set aside for those who are developing DNA diagnostic tools: "They had an interesting proposal that would not only develop the chip technology, but also the [readers], etc. They had an ambitious, broad-based technology."
Under the grant, the consortium will develop miniature, integrated devices for a wide variety of DNA diagnostics. Affymetrix ultimately envisions a hand-held device capable of accepting a patient sample, extracting DNA from the sample, amplifying the target DNA using established techniques, and analyzing the resulting data. Molecular Dynamics will design the hand-held reader, a product that may be several years from realization.
Meanwhile, engineers at the Hewlett-Packard Company are working alongside Affymetrix to develop and manufacture scanners that will be marketed in the near future. The chip and reader system will help researchers obtain and analyze data gathered from the GeneChip arrays. They are designed to give test results in only two to three hours, whereas standard gene sequencing tests can take a full day to complete.
Another collaborator on the DNA GeneChip products is the Genetics Institute, a Cambridge, Massachusetts, biopharmaceutical firm engaged in the development of drugs through recombinant DNA and other technologies. Genetics Institute is providing research funding for the development of novel assays using Affymetrix's GeneChip technology. The company hopes to apply the chip technology to their particular area of expertise, gene expression monitoring. Using the GeneChip arrays, Genetics Institute scientists can rapidly and simultaneously screen for the expression of a specified group of genes in both normal tissue and diseased cells, thus identifying potentially important therapeutic proteins.
Sequence of Progress
Just as photolithography has transformed the microelectronics industry, the microchip has the potential to transform scientific research. Fodor, Rava, and their colleagues devised a method in which light directs the simultaneous synthesis of tens to hundreds of thousands of different chemical compounds in precise locations on a solid support, the microchip. Affymetrix also developed a complementary technology that uses laser confocal fluorescence scanning to detect molecular binding events at different points on the array. Used together, these technologies make up a miniaturized test format that promises to transform scientific understanding of the molecular basis of human disease.
Here's how the chip is made: In the first of many cycles, the chip is coated with a nucleotide linked to a light-sensitive chemical "block" group that halts subsequent reactions. Light is directed through a photolithographic mask, illuminating specific grid squares on the chip and causing photo-deprotection, or the removal of the block group in those squares. The chip is then coated with another photo-protected nucleotide, which reacts with the exposed squares. The cycle continues: the chip is exposed to light through the next mask, which activates new grid sites for reaction with the subsequent photo-protected nucleotide. At each step, chemical coupling occurs at the places on the chip that have been illuminated in the preceding step.
Using combinatorial masking strategies, scientists can synthesize large numbers of compounds in a small number of steps. With light-directed, spatially addressable chemical synthesis technology, Affymetrix can, on a single chip, and in 32 chemical steps, construct an array of all 65,536 possible combinations of eight nucleotides, with each one in a known location. The position of sites within the array and the resulting compounds depend on the patterns of illumination and the sequence of reactants used.
In addition to the advantage of its small scale, this method yields another benefit: "A target molecule can now be incubated with the array, allowing the assay to be carried out against all members simultaneously," Fodor and colleagues reported in their paper published in Nature in 1993. Fluorescent labeling of the assay molecule reveals specific interactions with individual members of the matrix.
Right on target. Fluorescence image of a 256-octanucleotide array following hybridization.
Source: Affymetrix/PNAS
After the chemical array is assembled, the chip is mounted on a thermostatically regulated flow cell. The fluorescently tagged target molecules--single-stranded DNA, for example--are put into a solution. Probing the solution, the microchip searches for specific sequences. Bright fluorescent squares would show sequence matches, while dim ones would reveal single-base mismatches. According to Fodor, the matches could uncover genetic defects, such as the gene linked to cystic fibrosis. Analysis of both matches and mismatches could also reveal the complete nucleotide sequence in a set of DNA fragments.
DNA sequencing is one of the more promising applications of GeneChip technology. Conventional DNA sequencing methods are cumbersome and time consuming, requiring electrophoretic size separation of labeled DNA fragments. In searching for a tool to rapidly extract and analyze genetic information, Fodor and colleagues pioneered a new generation of gene sequencing technology they term "sequencing by hybridization," or SBH.
The SBH technique uses a microchip containing a group of short oligonucleotide probes of defined sequence, which are used to search for complementary sequences on a longer target strand of DNA. The target DNA sequence is then replicated using the hybridization pattern. In one test, a researcher or clinician could identify many complementary sites on target DNA and avoid the hundreds, perhaps thousands, of steps that might be required using conventional hybridization methods.
Fodor and his associates contend that this new sequencing technique will speed scientific investigations into human genetics and diagnostics, pathogen detection, and DNA molecular recognition. "Current sequencing methodologies [rely] on complex procedures and require substantial manual effort," the team reported in Nature. "Sequencing by hybridization has the potential for transforming many of the manual efforts into more efficient and automated formats."
And the technique should prove to be not only faster, but more accurate as well: matching hybridization patterns may help resolve sequencing ambiguities from standard gel techniques. Because single-base changes cause multiple changes in the hybridization pattern, oligonucleotide arrays could become a powerful tool for checking the accuracy of a previously identified DNA sequence or to scan for changes within a sequence.
Fingerprints and Mutations
The GeneChip system has the potential to speed diagnosis and refine treatments for diseases such as AIDS and cancer. An enhanced understanding of genetic influences on the human immunodeficiency virus--which may dictate how an AIDS patient responds to a drug therapy, for example--could transform the way the doctors use drugs to treat the disease. "One of the challenges in treating HIV," Rava explains, "is the viral genetic diversity that confers drug resistance. The objective of a GeneChip analysis of the HIV genome is to provide a genetic fingerprint of viral infection."
To obtain such a fingerprint, a clinician would use a DNA chip encoded with more than 4,000 probes designed to detect most known mutations of the viral gene. The virus is first separated from the patient's blood sample, then DNA is extracted and split into single strands. The strands are then broken into fragments and labeled with fluorescent dye. Probes on the DNA chip then bind to complementary sequences among the patient's DNA fragments. Next, a laser scans the chip, illuminating the resulting DNA matches and allowing researchers to reconstruct the genetic sequence of the virus and determine if, for example, it has a mutation leading to drug resistance.
"Pharmaceutical companies can use this [technology] in an experimental program to develop drugs," Berg explains. It would serve as a useful research tool to help them know how to combine drug compounds, as well as monitor changes that lead to drug resistance, he says.
Affymetrix is also building a DNA chip to test for mutations in the p53 gene, which plays a critical role in many types of cancer, including breast, lung, and colon cancer. This chip is targeted to researchers investigating the relationship between genetic mutations and the onset of cancer. The chips might also be used to monitor mutations in genes caused by external environmental agents, Rava says.
Other DNA chips will detect inherited diseases. One chip under development will be able to detect many of the 450 mutations linked to cystic fibrosis, a devastating childhood disease. The system can identify more mutations in the cystic fibrosis gene with more reliability than conventional screening methods.
The Next Generation
Although Affymetrix and partners are currently making the GeneChip system for use in most clinical diagnostic fields, they envision other applications as well--in forensics, industrial testing, agriculture, and biomedical research. "The range of uses is just extraordinary," claims Berg. "We've hardly scratched the surface of the potential of these chips."
GeneChip technology could play an important role in the emerging field of genomics, a new field in biomedical research aimed at clarifying the relationship between genetic sequences and human physiology. "With genomics," says Rava, "we make measurements at the DNA level to make determinations about the biological state of an organism. We hope to have a technology that will eventually enhance everyone's work in the genomics field."
"The Human Genome Project is catalyzing a revolution in the understanding of human disease," says Robert Lipshutz, director of advanced technology at Affymetrix. "Cost-effective tools, such as those we intend to develop under [the Department of Commerce] grant, are needed to apply these advances broadly in the healthcare field."
According to Steve C. Clark, senior vice president for discovery research at the Genetics Institute, many of the approximately 70,000 genes in the human genome have already been identified, but the function of most of the genes is unknown. "Knowing a gene's identity, or DNA sequence, often contributes little to understanding its biological function," Clark explains. Scientists at the Genetics Institute hope their research using the DNA chips will lead to the discovery of new drugs. "Discerning how a gene is regulated and what biological role it may play in disease provides a basis for designing important new drugs," says Clark.
The emerging DNA chip technology shows incredible promise, but its creators see drawbacks as well, mainly in the unknown factors that lie in the path toward any new technology. "The biggest drawback is that, with any new technology in development, there are unforeseen challenges," Rava concedes.
For now, the technology's advantages seem to vastly outweigh any disadvantages. Bolstered by that conviction--and the Department of Commerce grant--the key players in DNA microchip technology are now refining techniques for the manufacture of the first GeneChip arrays targeted to users in the research marketplace. Affymetrix has come a long way toward realizing this important application in a relatively short time: the company first identified the opportunity to use DNA chip technology in the areas of research and diagnostics just 18 months ago, and, if all goes according to plan, the company will ship the first chips to laboratories later this year.
Jennifer Medlin
Jennifer Medlin is a freelance writer in Cary, North Carolina.
Suggested Reading
Fodor SPA, Rava RP, Huang XC, Pease AC, Holmes CP, Adams CL. Multiplexed biochemical assays with biological chips. Nature 364:555-556 (1993).
Fodor SPA, Read JL, Pirrung MC, Stryer L, Lu AT, Solas D. Light-directed, spatially addressable parallel chemical synthesis. Science 251: 767-773 (1991)
Gordon EM, Barrett RW, Dower WJ, Fodor SPA, Gallop MA. Applications of cobinatorial technologies to drug discovery. J Med Chem 37(10):1385-1401 (1994).
Pease AC, Solas D, Sullivan EJ, Cronin MT, Holmes CP, Fodor SPA. Light-generated oligonucleotide arrays for rapid DNA sequence analysis. Proc Natl Acad Sci USA 91:5022-5026 (1994). |
Last Update: April 8, 1998