IVD Technology Magazine
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Originally published November, 1998

DNA-chip technologies

Part 2: State-of-the-art and competing technologies

Cliff Henke

DNA microarray product development is proceeding with one underlying principle: fast is not fast enough. This is the second of a three-part series on the emerging technology. If you haven't already done so, you might like to begin with Part 1 of this series.

Also in this article:

• Cepheid bridges the sample-volume gap
• Microarray technology development at Genometrix
• Caliper harnesses electrokinetics
• Vysis prepares to launch Genosensor array technology
• Microtechnology development at PE Applied Biosystems

For those who marvel at how quickly DNA chips have burst upon the scene, prepare to be astonished further. Most companies report rapid progress, and suggest that as more is learned and applied, the pace of development in all fields of genetic-related diagnostics and therapeutics—including DNA microarrays—will continue to accelerate.

Labchip by Caliper Technologies (Palo Alto, CA), showing photolithographically etched fluid channels (in red). Channels are typically 70 µm wide and 10 µm deep. Photo Courtesy Caliper Technologies

There is no better example of this than the Human Genome Project (HGP). When the project was launched in 1990, government officials viewed it as perhaps a 20-year project. Proponents compared the vision of the project to that of America's goal of landing a man on the moon in the 1960s—but conducted with international cooperation and over an even greater period. Almost as soon as the HGP was launched, the date for completion was revised to 2005, then to 2003, thanks to developments in computerization, telecommunications, and molecular biology. Today, because of DNA chips and related developments, some officials now believe that the HGP can be completed by the end of this decade—three years ahead of even the revised schedules. In turn, as more is learned in this massive effort, it will accelerate DNA-chip development even more, observers say.

This is the second installment of a three-part series on DNA-chip technologies. The first reviewed the theoretical underpinnings of the field and examined the market forces driving product development. This installment will look at the state of the art and the various competing technologies in this embryonic field. The final article will cover the challenges to commercialization facing companies engaged in this new marketplace as well as prospective near- and long-term applications for these technologies.

Fast-Moving State of the Art

"The outlook is changing fast," says Deepak Thakkar, manager of microarray products at Genometrix (Woodlands, TX). "As an industry, we have largely been in a hibernation period, but in the past 18 months we have introduced a lot of highly targeted products based on very specific needs."

As an indicator of how rapidly the pace of development has proceeded, consider that the first DNA chip, by Affymetrix (Santa Clara, CA), was introduced only two years ago. Today two dozen or more firms are actively engaged in developing microarray technologies, and many others are developing related technologies for sample preparation and analysis.

"Until recently, most of this development work has been centered on what I would broadly call detection," says Peter Wilding, PhD, director of clinical chemistry and professor of pathology and laboratory medicine at the University of Pennsylvania (Philadelphia). "The vast majority of the products that have been introduced, however, are sold for highly specialized applications, usually in research-laboratory or drug-development applications."

Such applications usually involve some genomic-related problem. For example, the gene p450 and its eight different expressions, or mutations, have been linked to various cancers. Researchers are examining exactly which forms have the closest link to these cancers. In turn, drug developers want rapid genomic results so they can quickly determine the effectiveness of candidate therapies.

The reason that research-laboratory and drug-development applications are being developed is simple: that's where the money is. Governments, drug companies, and universities have poured billions of dollars into developing DNA chips for applications related to their agendas.

Take governments. The Defense Department's Advanced Research Projects Agency (DARPA) and the Commerce Department's Advanced Technology Program (ATP) at the National Institute of Standards and Technology (NIST) have invested millions of dollars as seed money for developing both various kinds of chips and their basic manufacturing technologies. "All the major players in this field have received research money from ATP," says Uwe Müller, director of advanced technology at Vysis, Inc. (Downers Grove, IL). "Stan Abramowitz and NIST should get a lot of credit. The ATP is a government program that is actually working, even with minute funding levels, and its support of this technology is the envy of the world."

DARPA has contributed millions more. In its case, DARPA funds projects for "dual use," meaning they must have both civilian and military applications. The prospective military use of DNA-chip technologies is for advanced detection of battlefield biological and chemical weapons.

Private-sector sources of funding have followed with billions more, initially from venture capitalists, and later from strategic partnerships, institutional investors, and public stock offerings. Yet these sources of funding have made pursuit of short-term financial objectives even more urgent, several observers point out.

As an example of this, Müller recalls: "Before Vysis was spun off from Amoco Technology, we were working on a food diagnostic application for E. coli testing; but we put aside that project. We realized that the food processing industry is very competitive, and the low profit margins for the tests used in that industry would not bear the heavy costs of research and development for DNA chips."

Today, such economic inducements are readily apparent. Almost every major manufacturer of DNA chips has a strategic relationship with a major drug company, taking advantage of the eagerness of pharmaceutical manufacturers to find ways to screen potential drugs faster and more accurately. These relationships usually consist of "early access partnerships," in the words of Lewis Gruber, president of Hyseq (Sunnyvale, CA). "This means that partners have access to a company's technology, often for specific applications only, before others do."

"If these technologies succeed in giving pharmaceutical companies the ability to screen drugs quickly and accurately, the payback will be tremendous," agrees Thakkar. "Only one drug in 10,000 succeeds in the marketplace and it takes 12 to 15 years to develop each new one. If these technologies can reduce the length of development time to eight years, that translates into significant savings for these companies."

An example of such corporate relationships is the set of strategic agreements announced earlier this year by Affymetrix and Eos Biotechnology, Inc. (South San Francisco, CA). The agreements allow Eos broad access to Affymetrix's custom and standard GeneChip expression chips for Eos's molecular genomics research efforts in specific fields of cancer, inflammation, and cardiovascular disease.

However, some analysts, including Peter Wilding, believe that such drug and research partnerships are stifling innovation that could lead to larger markets. "If the use of chips remains an activity that is confined to research laboratories—because you need their equipment to prepare samples and do the analysis—then the markets for DNA chips will be limited," he says.

Such thoughts are leading companies into the next phase of microarray development, which is already well under way. A number of companies are developing technologies that use immunoassay reagents or amplification techniques such as polymerase chain reaction (PCR) to prepare samples right on the DNA chip. Companies are also exploring technologies for conducting sample analysis on the same chip and outputting results to a single small instrument. Taking small sample amounts and moving them through the various stages of sample preparation, detection, and analysis of test results is being made possible by advances in capillary electrophoresis. Using electrical charges and micro- and nanoscale structures, researchers have successfully demonstrated that it is possible to perform all of these stages on a single DNA-chip system.

"We think we can have such a product in the marketplace by the third quarter of 1999," says Cris McReynolds, director of business development for Cepheid (Sunnyvale, CA), a maker of sample-preparation and analysis equipment designed to be integrated with DNA-chip technologies. Others echo his comments regarding their firms' product development timetables.

Types of DNA Chips

There are three basic types of DNA chips. The first and oldest is the sequencing chip. This is also the type most commonly discussed in popular articles about this technology. With sequencing chips, such as those initially produced by Affymetrix or Hyseq, segments of DNA (usually 20 bases long) are placed in a microarray. Target samples are then introduced to the chip and the segment that the sample "sticks to" (or hybridizes with) determines the result. This design is called sequencing by hybridization (SBH), and is both an industry term and an intellectual property of Hyseq, says Gruber. Many other companies are now producing sequencing chips, most using the SBH approach. But whatever their technique, such products are intended to determine the DNA sequence of the sample.

The second variety of DNA chips is known as the expression chip. These are designed to determine the degree of expression of a certain genetic sequence by measuring the rate or amount of messenger ribonucleic acid being produced by the target gene. This is done by creating chips with a specific set of base pairs (as opposed to sequencing chips, wherein every possible base-pair combination is arrayed). Results are then compared to a reference or control, and the degree of change is noted. These chips are useful in diagnosing and treating diseases linked to particular genetic expressions, such as some forms of cancer. Vysis and Synteni (Fremont, CA) are two companies engaged in marketing expression-chip-based products and services.

The third type of chip is devoted to comparative genomic hybridization. It is designed to help clinicians determine the relative amount of a given genetic sequence in a particular patient. "A certain amount of unusual genetic expression is normal, but it becomes a cancer out of control only when the level of expression reaches a dangerous level. As an extreme example, many breast cancer tumors—particularly at the end stages of the disease—are so violently aberrated genomically that they don't even have 23 chromosomes anymore," Müller points out. "This type of chip is designed to look at the level of aberration." This is usually done by using a healthy tissue sample as a reference and comparing it with a sample from the diseased tumor.

Design Goals

In the first installment of this series we reviewed the types of technologies companies are using to manufacture their products. While those basic technologies have not changed, rapid progress is being made in all of them to increase density (the number of arrays per chip) for the next generation of products in development. Whether via robotic deposition or microlithography, development is proceeding in pursuit of two basic design goals: increased sensitivity and reliability, and systems integration.

In the case of the former, the "holy grail is to get better discrimination," says Lance Fors, president of Third Wave Technologies (Madison, WI). "The key to better discrimination is improved signal-to-noise ratio." This is especially important since so many DNA chips depend on PCR-based amplification of the sample, which can undermine discrimination. "A challenge facing us is to find a tumor cell amidst a thousand healthy cells in a biopsied sample; it's like finding a needle in a haystack," he says.

Third Wave and other companies are working to reduce needed sample size by decreasing the amount of reference sample, thus reducing the amount of PCR amplification needed. The reduction of the amounts needed is being accomplished through advances in microfluidic technology. For example, capillary electrophoresis breakthroughs and nanoscale fabrication development have enabled companies to reduce needed reference and target sample sizes to microliter and picoliter scales.

In addition, says Wilding, piezoelectric charges applied to these tiny capillary tubes can move samples from preparation to reaction on a single chip. This achievement has laid the groundwork for the second goal, systems integration. "To do this you must have a microfluidics platform," he says.

"Systems integration is no longer a pipe dream," Wilding continues. "Five years ago an industry survey identified a dozen or so companies developing chips, nearly all of which were involved in detection applications. Three were involved in reactions, and none were doing sample preparation. Today nearly everyone is involved in all of these applications, and all have recognized the importance of an integrated approach."

An example of this work is the collaboration among the U.S. Department of Energy's Argonne National Laboratory, Engelhard Institute in Russia, Motorola, and the Packard Instrument Co. This consortium is working on a microarray-based system that would perform sample preparation (using PCR amplification) and reaction on a single chip and within a single analytical device.

"I think you'll see integrated systems that will address some key markets in clinical chemistry sometime in 1999," predicts Thakkar. He agrees with Wilding that these will be integrated systems designed for ease of use.

Ultimately, within the next 10 years, Wilding and others predict, DNA chips and integrated processing will penetrate the emerging point-of-care market. To achieve this, however, myriad commercialization hurdles must be overcome.

Conclusion

DNA-chip development is proceeding at a pace so rapid that it surprises even the most optimistic members of this fast-emerging industry. "The question 'Will DNA chips succeed?' is a dumb one," notes Wilding. "The only real questions are how they will develop and how quickly."

As companies look to market products outside the research-laboratory and drug-development environments, they will face enormous regulatory and market challenges. Chief among the regulatory challenges is developing a set of industry standards for quality assurance. Today each company has its own. "This will involve some of the big companies like Affymetrix taking the lead, and it will require a real partnership between FDA and all the key players in the industry," says Thakkar.

The biggest marketplace hurdle will be to introduce products at prices that are competitive with existing technologies, says Wilding. "In today's reimbursement climate for health care, products must be better as well as cheaper," he points out. These are issues that will be discussed further in the third and final installment of this series, which will discuss future developments.

Cliff Henke is a freelance writer based in Southern California.

Cepheid bridges the sample-volume gap

Kurt Petersen

Regardless of the details of any particular technique, diagnostic applications of DNA-chip technologies must contend with one overriding and frequently overlooked requirement: they must be sensitive to low-concentration target analytes. Modern DNA diagnostic assays face mounting demands to detect organisms or DNA mutations at very low concentrations, often less than 100 copies per ml, in raw biological samples such as blood or urine.

Such sensitivity requirements set fundamental physical limitations on the minimum quantities of the starting sample. For example, polymerase chain reaction (PCR) is usually considered capable of detecting as few as 20 copies of a target in any given, purified, biological sample. But if the required assay sensitivity is 100 copies per ml, then a minimum of 200 µl of the original sample must be processed to ensure statistical confidence in the result of the assay. The diagnostic requirement for such large sample volumes is deeply at odds with the headlong dash toward nanoliter- and microliter-volume sample sizes that has been the operational standard for some DNA-chip research efforts. In some phases of diagnostic protocols, smaller is not always better.

Cepheid's Smart Cycler system employs a Windows-driven user interface to assign and monitor a unique thermal cycling and real-time detection protocol to each reaction site, enabling parallel processing of diverse nucleic acid analyses. Key to the system is a disposable sample-preparation cartridge with microfabricated components (inset). Photos Courtesy Cepheid, Inc.

Cepheid, Inc., is committed to developing and implementing fluidic processing technologies that bridge the gap between the demand for such "large" sample volumes and the world of microstructures and microarrays. Cepheid is attacking this problem on two fronts. First, the company has developed the only available thermal-cycling technology for performing fast PCR on large sample volumes. The Cepheid I-Core technology is capable of attaining less-than-30-second PCR cycles for volumes as large as 100 µl, while simultaneously detecting and quantifying fluorescent DNA products in real time.

More than 90% of the customers and users of DNA hybridization chips require a nucleic acid amplification step (typically PCR or RT-PCR) prior to hybridization. In fact, PCR is becoming increasingly recognized as the final step in the sample preparation process, wherein the raw biological sample is prepared for the DNA identification and detection technique. Fast PCR on large sample volumes is a first step toward achieving practical, high-sensitivity, diagnostic DNA assays. I-Core modules can serve as the front-end for generating sufficient quantities of DNA amplicons for use with DNA hybridization chips, as well as by other sequencing and analytical tools.

On a second front, Cepheid is addressing the problem of efficiently miniaturizing the next "upstream" fluid processes in nucleic acid assays: nucleic acid purification and concentration. In this context, it must be realized that the concept of a DNA chip is not limited to planar arrays of hybridization sites. Cepheid has developed a micromachined, microfluidic technology in which high-surface-area structures are created in silicon substrates. Such structures can be designed and organized into well-defined arrays that optimize fluid flow and liquid-surface interaction. Although the internal volume of such a chip may be small (several µl), its array of micromachined columns can capture dissolved nucleic acid as it flows through the chip.

Chips incorporating such microstructures can efficiently capture DNA, either specifically or nonspecifically, and concentrate and elute the captured DNA into small volumes. In this way, large-volume fluid samples can be flowed through the chip; the chip is continuously capturing and concentrating the nucleic acid in the fluid sample; and the nucleic acid is later eluted into a much smaller volume. In early development efforts, capture efficiencies greater than 50% and concentration improvements approaching 20* have been achieved.

Cepheid is applying advanced chip-based technologies to one of the most difficult aspects of practical DNA diagnostic assay realization: sample preparation. Although hybridization chips are not required for every DNA assay, sample preparation is a universal requirement. Novel chips for nucleic acid purification and concentration, combined with fast PCR, will eventually enable diagnostic assays to be transferred out of research and clinical laboratories into miniaturized, cartridge-based formats. These developments will radically alter the future implementation and application of DNA diagnostic assays. By starting with the fundamental requirements of such assays, Cepheid is pioneering the inevitable trend toward point-of-use DNA diagnostics.

Kurt Petersen is president of Cepheid, Inc. (Sunnyvale, CA).

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Microarray technology development at Genometrix

Deepak Thakkar

Genometrix is in the business of developing instrumentation and consumables for high-throughput, low-cost microarray systems. The company has developed several platforms that find applications in gene-expression analysis, mutation screening (population-wide genetics), and drug discovery. Genometrix is working with a number of research groups, both academic and industrial, to validate and establish these platforms.

The company has focused its attention in the direction of developing low-cost, medium-density tools that have the potential to screen a large number of samples (tens of thousands of tissues or different drug compounds) in parallel. The drug-discovery chips are intended for high-volume use and have a base price starting from $10 to $100 per chip, depending on the number of elements and volume of chips ordered. The company has also developed an automated workstation for sample processing and automated chip hybridization. This prototype workstation will allow for mRNA isolation, automated PCR, hybridization, and chip detection at the rate of up to 1000 samples per day.

Genometrix's current manufacturing capabilities enable the company to perform routine production of low- to medium-density chips (up to 256 elements per array) at a rate of up to 10 million chips per year. The capillary-based array manufacturing facility is located in a Class 100 cleanroom area. The array manufacturing process is flexible, and therefore has the capacity to accommodate rapid customization of its content. The company's high volume of production will facilitate population-wide screening and drug discovery processes, including the manufacture of arrays for use in clinical trials and routine diagnostics.

The chips are available in a variety of formats, including 8-, 18-, and 96-well glass slides. The chips have been developed in such a way that the volume of sample required for target hybridizations is less than 50 µl per well, allowing complex assays to be performed on 50,000 to 75,000 cells. This low sample volume is suitable for high-throughput screening using cell cultures in a 96-well format.

Deepak Thakkar, PhD, is product manager for microarrays at Genometrix, Inc. (Woodlands, TX).

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Caliper harnesses electrokinetics

Anne R. Kopf-Sill

The focus of Caliper Technologies Corp. is to develop laboratory-on-a-chip technologies and products. Such technologies enable researchers to move minute amounts of fluids in tiny channels on integrated microfluidic devices, in order to perform a wide variety of biochemical and cell-based assays. The advantages of this approach are speed of analysis; small reagent and sample consumption; and high-quality, computer-controlled data generation. In effect, Caliper intends to do for the biochemical lab what electronic chips have done for information technology. Smaller, more-integrated functions lead to more-powerful capabilities.

Microfluidics and Fluid Motion

Caliper's microfluidic devices will be marketed under the Labchip trademark. Labchip fluid channels are created photolithographically in either glass or plastic substrates, and are typically 70 µm wide and 10 µm deep. The fluid network can be changed with each device design.

The Labchip technology is based on electrokinetic fluid movement, which combines electrophoresis and electroosmosis. Using electrophoresis, molecules in an electric field can be made to move toward one or another electrode, depending on the electrical charge of the molecules. Using electroosmosis, on the other hand, bulk fluids in a channel or capillary can be moved by applying an electric field along the length of the channel. The fluids involved can be any aqueous-based buffer.

Figure 1. Top-down view of a Labchip with a four-port cross-structure. The channels are filled with buffer (black) and a red dye. Left (a), the voltage applied is high at the upper port and low at the bottom port, causing the buffer and dye to move from top to bottom and fill the intersection. Right (b), the applied voltage is high on the right and low on the left, causing the buffer to move from left to right, taking with it the dye at the intersection. Photos Courtesy Caliper Technologies.

Electrokinetic control permits researchers to move fluids and other materials around in a network of fluid channels without the use of external pumps or valves. The application of computer-controlled electrical signals offers exquisite control of fluid motion and reaction timing. For example, the system can be instructed to mix two reagents, wait a fixed time before introducing a third reagent, incubate, and then separate the reaction products downstream.

Figure 2. A Labchip with a four-port cross-structure using electrokinetic fluid movement to make variable-volume injections. Top (a), voltages are normally applied so that dye moves from right to top and buffer moves from bottom to left. Middle (b), voltage is applied from right to left and some of the dye is injected into the left-hand channel. Bottom (c), two dye plugs are injected into the channel, with differing sizes determined by the time of the second voltage state.

Examples of electrokinetic fluid motion are shown in Figures 1–3. Figure 1 shows the capability of an electrokinetic system to move a measured amount of fluid (defined by the size of the channels in the intersection) in a desired direction. In this instance, the volume of dye injected is 40 pl, which is about one-millionth the size of a drop of liquid. Figure 2 shows the system's ability to make variable-volume injections, with the amount determined by the length of time that a voltage is applied. This scheme enables researchers to vary the volumes of liquids while an assay is being performed. In this use, the intersection acts like a valve, but has no moving parts, no leakage, and very fast response times. Figure 3 shows a fixed-volume injection and chemical separation. The inlet and outlet are offset from one another, which allows a much larger volume of dye to be injected, in this case 300 pl.

Figure 3. Fixed-volume injection and chemical separation, using a dye mixture of two different dyes with different charges. The front dye is neutral and moves with the buffer; the second dye is negatively charged and is retarded by its attraction to the positive electrode on the right-hand side.

Biochemical Assays

Caliper is developing a wide variety of assays, with the ultimate goal of putting virtually all the assays performed in a biochemistry lab onto Labchip microfluidic systems. To study enzyme inhibition, for instance, the substrate, enzyme, and inhibitors are placed in three separate wells on a microfluidic device. Each of these is paired with a buffer well, making it possible to control the concentration of each reagent without affecting the concentration of the others. In use, substrate only is pumped in to obtain a substrate background reading; then substrate and enzyme are pumped in together to obtain the product signal from a fluorogenic substrate; and finally inhibitor is added to determine the percent of inhibition for that particular system.

The Labchip system can also be used to study enzyme kinetics. The concentration of the substrate can be varied easily over a wide dynamic range and the product concentration measured. Enzyme parameters such as K_m can be determined in a few minutes.

By filling the microfluidic device channels with a sieving matrix, the Labchip system can be used to size and separate DNA fragments. Because the length of sample applied to the separation column is very short (just the channel width) the separation of fragments is extremely rapid, in seconds rather than minutes on a slab gel. Single base-pair resolution out to approximately 400 bases is achievable in 5 minutes. Using a simple Labchip design, polymerase chain reaction (PCR) can be performed on a whole blood sample and the resulting product peak-separated in a sieving matrix on the same chip.

Caliper has generated data on cell-based assays. Cells are used as a reagent in one of the wells, and agonist from another well is added into a central reaction and detection channel. A fluorescent signal is detected downstream, after each of the passing cells has had a 10-second exposure to the compound. Assays tested have included intracellular calcium flux with either ionomycin or UTP activation. Tests have shown the system can generate binding and kinetic data for the cellular responses.

Sample and Compound Accession

Since the samples and compounds that people typically want to test are in the macro world, Caliper has developed a world-to-chip interface as a key element of its technology. The Labchip is attached to a glass capillary and electrode; the capillary is then dipped into the sample or reagent in a microwell plate, an electric field is applied, and the samples are electrokinetically brought into the Labchip microfluidic system. This system has been used in enzyme inhibition studies, for example, in which a new test compound is loaded into the chip every 5 to 10 seconds.

Comparison to Array-Based Systems

Caliper thinks of its devices as analogous to microprocessors in the electronics business. The fluid network can be designed differently for each type of assay; the timing of reactions and concentrations of reagents can be changed for a given device design; the reagents on the device can be changed from run to run; and the samples or compounds can be changed with each test. Although some DNA-array systems can make hundreds or thousands of DNA determinations at one time, they are limited to DNA hybridization assays and to a single sample across the device. By comparison, the Labchip system is distinguished by its flexibility in performing various types of assays and its speed of sample and reagent acquisition.

Business Plan

Building on the company's core technologies in microfluidics, electronics, assay development, and device manufacturing, Caliper has established itself as the leader in the laboratory-on-a-chip field. The company's general strategy is to focus on the design, development, and fabrication of Labchip microfluidic devices and systems, collaborating with other companies to sell and use the chips and instruments in their area of specialization. The company is collaborating with pharmaceutical firms to develop high-throughput microfluidic devices for screening of drug compounds. Another major collaboration involves Hewlett-Packard, which will market Labchip microfluidic systems into the research market. In the next few years, Caliper expects to develop other partnerships in the drug discovery arena as well as partnerships to serve the genomics and diagnostic markets.

Caliper believes that Labchip microfluidic technology can fundamentally change the nature of biochemical experimentation and the process of biological discovery. The ability to exercise computer control over all parameters of an assay will make it possible for even complicated assays to be performed in almost any laboratory. And the rapidity of such testing, combined with reduced use of reagents, will expand the number of tests that a laboratory will be able and willing to perform in a day. The volumes of experimental data produced on such systems may signal the arrival of the information age in the business of biological research.

Anne R. Kopf-Sill is manager of the microfluidics and advanced technology group at Caliper Technologies Corp. (Palo Alto, CA).

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Vysis prepares to launch Genosensor array technology

Uwe Müller

The amplification and overexpression of genes are known to play important roles in the development of many types of tumors and prenatal diseases. For use as a research tool—with plans for subsequent use in routine diagnostic applications—Vysis has developed a DNA-array technology that can measure changes in the number of copies of specific DNA or mRNA sequences, using a multicolor, comparative hybridization approach.

In genomic research applications, the aim of the company's technology is to measure gene amplifications, gene deletions, or unbalanced chromosome translocations. The company's first product, called the Genosensor system, consists of DNA arrays, instrumentation, and analysis software that will give researchers a powerful new tool in cancer research. The system is scheduled for commercial introduction in 1999.

Composite image from a Vysis Amplionc array chip cohybridized with human reference DNA (red) and brain tumor DNA (green), with a blue counterstain.

Computed ratio of green/red oncogene targets from a Vysis array, indicating amplification of oncogenes EGFR, MDM2, and CDK4. Controls are on right end of graph. Photo and Illustration Courtesy Vysis, Inc.

Genosensor arrays include the company's trademarked Amplionc assay, which contains all genetic regions so far reported to be associated with tumor formation through amplification at the genome level. DNA clones comprising the desired target sequences are arrayed on a coated glass chip in multiple target spots of 100 to 200 µm in diameter. The assay involves the labeling of tumor DNA with a green fluorophor, which is then mixed with an equal amount of whole genomic reference DNA that is labeled with a red fluorophor. To suppress repeat sequences, the array is cohybridized in the presence of Cot-1 DNA. Target spots are counterstained with a blue fluorophor for identification. After hybridization, the array is imaged by the Genosensor reader, a multicolor, large-field fluorescence imaging system. The reader's software analyzes each target area and calculates the ratio of green to red fluorescent emissions, which indicates whether a specific sequence in the sample DNA is deleted, amplified, or present in a normal amount relative to all other targets.

In applications related to prenatal diagnosis, Vysis is developing an array that can quantitate all the DNA sequences that are known to be associated with specific disease syndromes. Most of these are caused by relatively small deletions of chromosomal regions that are below the resolution of traditional cytogenetic methodologies and typically go undetected. The ultimate goal is an array that can screen the complete human genome for such deletions, with a 10- to 100-fold increase in sensitivity at a significant reduction in assay time and cost.

The same Genosensor system can also be used to measure differential expression rates between reference and test tissues. Vysis is developing specific expression arrays that contain all known oncogenes, tumor-suppressor genes, and other disease-related genes of interest. Immediate research applications are aimed at establishing a correlation between measurable molecular changes and important clinical parameters, such as tumor progression, response to therapy, and disease outcome.

Uwe Müller is director of advanced technology at Vysis, Inc. (Downers Grove, IL).

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Microtechnology development at PE Applied Biosystems

Evelyn Shulakoff

Miniaturization for life science research applications is a major research and development program at PE Applied Biosystems, with funding of approximately $10 million annually.

Because no single format meets the broad needs of life science researchers and pharmaceutical developers, the company has several microformats in development. These include universal hybridization chip formats and projects for the miniaturization of entire systems, which could include amplification, electrophoresis, and sample preparation. PE Applied Biosystems is also developing fluid-handling robotics and tools to deliver the submicroliter volumes of reagents and samples for these microsystems in a precise and controlled manner.

The ever-shrinking size and sample-volume requirements of DNA analysis: from left, standard microamplification tubes (sample ~50 µl); a standard 96-well microplate (~25 µl); and products by PE Applied Biosystems, a 96-well microcard (1 µl), a miniaturized 49-well array (1 µl), and a 1000-well array (30 nl), also shown in inset. Photos Courtesy PE Applied Biosystems.

The company intends to apply its substantial portfolio of intellectual property, as well as those from collaborations, to its microtechnology program. The initial phases of the microtechnology projects are managed through the PE Applied Biosystems science and technology group, the company's breeding ground for next-generation technologies and applications. The mission of this group is to assess new technologies and business opportunities and provide the initial development of key capabilities.

Funding and Collaborations

Federal grants fund a significant portion of the company's research. These include a grant from the National Institute for Standards and Technology for the development of microgenetics employing polymerase chain reaction (PCR), electrophoresis, and hybridization arrays, and a Defense Advanced Research Projects Agency grant to develop microfluidics and fluidic modeling for microtechnology.

In 1997, Perkin-Elmer and HySeq, Inc. (Sunnyvale, CA), entered into a strategic partnership to further develop HySeq's proprietary DNA Hychip module, which is a universal DNA hybridization chip technology.

In 1998, PE Applied Biosystems expanded its microtechnology research program through a collaboration with Aclara Biosciences, Inc. (formerly Soane Biosciences, Inc.; Hayward, CA), for the optimization of electrophoresis in microchannel arrays.

Universal Hybridization Chips

Unlike the approaches of other array developers, PE Applied Biosystems expects to offer a universal hybridization chip technology that can be used for a multitude of genetic analysis applications—from gene discovery to genomic pharmaceutical development applications. The solution chemistry will provide the specificity of the test by employing technologies such as the company's proprietary PCR and oligonucleotide ligation assay technology.

For example, one universal chip project in progress is being done in collaboration with HySeq, Inc. Perkin-Elmer and HySeq entered into a strategic partnership to further develop HySeq's proprietary DNA Hychip module. This partnership is expected to accelerate the development and broaden the availability of a unique set of proprietary technologies from both companies that will advance the applications and commercial benefits of DNA sequencing arrays.

Chip Microsystems

PE Applied Biosystems also has many microtechnology projects in progress. System miniaturization is an enabling technology that would allow greater and faster throughput (simultaneous analysis of many more samples) at reduced cost (using less reagents) than is currently possible. For example, the company has developed a prototype of the Cytokine Card, a microcard that is the thickness of a credit card and contains 96 miniaturized, 1-µl reaction wells that contain Taqman reagents. The 50-µl volume of a standard PCR amplification is thus reduced to 1 µl in the Cytokine Card (see Table I). The microcard is used with PE Applied Biosystems' ABI Prism 7700 sequence detection system. The card enables batch-mode cytokine profiling for pharmaceutical drug development. Cytokine targets are common indicators of inflammation, infection, or toxicity. One can determine if the candidate drug causes inflammation or toxic responses by measuring the increase or decrease in cytokine expression after exposure to a candidate drug.

Table I. Progression of increasing number of analyses per run, and decreasing sample volumes required, by employing PE Applied Biosystems' microtechnology.

The primers and probes of 24 different cytokines plus an internal control come preloaded and dried down in the microcard's reaction wells. The user only needs to add sample plus reaction mix to one spot on the microcard and insert it into the ABI Prism 7700 sequence detection system to obtain results rapidly. The microcard is discarded after use. The microcard will provide higher throughput, user convenience, and a lower-cost screening test for pharmaceutical drug development. The microcard provides an example of the company's direction in microtechnology.

The ABI Prism 7700 sequence detection system (a combination thermal cycler and real-time detection system) provides batch-mode screening of samples for gene expression, allelic discrimination (such as used in population studies), and for the identification of specific sequences (such as sequence anomalies or microbial identification for research purposes). The system employs the 5' nuclease assay—a PCR technique that uses special fluorescently labeled probes. The microsystem offers real-time monitoring of the PCR amplification products (i.e., detects products as they are formed) in each of the microcard's 96 reaction chambers.

Impact of Microtechnology Projects

The microtechnology programs at PE Applied Biosystems are intended to provide substantial benefits to life science researchers such as throughput, speed, a high level of automation, cost savings, convenience, and ease of use compared to what is currently available, as well as provide new capabilities. The company offers complete systems, which, for chips, are expected to include instruments and data-analysis software that work in concert with reliable chemistry methods. In addition, PE Applied Biosystems provides reagents and protocols as well as training, service, and technical support. In combination, these products and services deliver total solutions to life science researchers for their ultimate success.

Many of the microprojects under way at PE Applied Biosystems are expected to result in prepackaged applications, thus making the sophisticated analyses of today easier and more routine tomorrow. The company expects that these systems will expand current markets by enabling more scientists to perform the studies.

The combination of benefits from the company's microtechnology projects is expected to accelerate research in all of PE Applied Biosystems' markets, which include basic research, genome projects, pharmaceuticals and molecular medicine, agriculture, microbial identification in food and the environment, and forensics.

Evelyn Shulakoff is science writer at PE Applied Biosystems (Foster City, CA).

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Continue to Part III.