|DNA and BioChips: 1
THE MAKING OF A MICROARRAY
When prepared properly, DNA chips and microarrays permit researchers to gather more data in less time. Here's a guide to the
steps in making chips and the products that simplify the process.
|BY PETER GWYNNE AND GARY HEEBNER
American Society for Biochemistry and Molecular Biology
Lab Products, Inc.
LION Bioscience, AG [Germany]
LION Bioscience [USA]
Packard Biochip Technologies
|•||An Expanding Business|
|•||The Big Bottleneck|
|•||Two Types of Chips|
|•||The Basic Steps|
|•||Onto the Chip|
|•||Labeling, Scanning, and Handling Data|
|•||From Gene Expression to SNPs|
|•||Strategy for SNPs|
The companies in this article were selected at random. Their inclusion in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
In just over a decade since their conception, DNA chips and microarrays have proven their value in genomics research and have forever changed the way in which molecular biologists approach their research. The rush to capitalize on information produced by the sequencing of the human genome is well under way, with researchers from many disciplines already acknowledging the power of these miniature laboratories on a slide. These early adopters have begun to reap the basic benefits from DNA microarrays: more data in less time.
DNA chips and microarrays allow the simultaneous examination of thousands of strands of DNA. At times, indeed, they create overpowering amounts of data. Thus the utility of these new laboratory tools depends on strong support from information technology. Fortunately advances in that sector have enabled the development of the powerful computer software programs necessary to make sense of such large volumes of data.
Potential users see huge promise in the technology. "The microarray market is not mature yet," says Siobhan Pickett, product line manager, array technology for Axon Instruments, Inc. "What's happening is that the early adopters and innovators are now moving into a mature phase in which they have the technology that's helping them to move forward to the next stages of their research. The market will grow in the near future as microarrays become a much more standardized technology in many types of life science laboratory, like gel electrophoresis and DNA sequencing."
The market already appeals to a broad range of consumers. "We originally assumed that our customers would be predominantly in the biopharmaceutical area," says Doug Amorese, section manager for biochemistry and chemistry in Agilent Technologies, Inc.'s Bio Research Solutions division. "We've been very pleasantly surprised by the demand in the academic and agricultural areas. This technology is being used across the board as it has matured to deliver lower costs and better product offerings."
John Burzcak, vice president for development of genomics at Amersham Biosciences (formerly Amersham Pharmacia Biotech), echoes that thought. "Pharmaceutical and life science companies have strongly embraced microarray technology," he says. "In the academic environment, microarray capabilities have been established in core laboratories that service individual investigators. As the technology matures, investigators' laboratories are clearly purchasing the tools for postspotting work, such as microarray hybridization reagents, kits, and instruments, as well as microarray scanners."
Further development of microarraying, and particularly of methods for interpreting the information that the method produces, will expand the technology's utility. "Now that the technique is developed you can optimize it," explains Horst Donner, head of R&D microarrays at German firm MWG Biotech AG. "However, it's not the technology but making sense of the data that will open the way to more use of microarrays. I'm convinced that the technology will soon find clinical applications."
Another factor essential to expansion of the industry is standardization of bioinformatics and many other aspects of microarray technology. Vendors of microarray systems and components face a situation similar to that encountered by computer companies 20 years ago, when they decided to move away from proprietary technologies to open systems. "The needs and interests of the scientific community will drive standardization," says Axon's Pickett. "But they are also looking for guidelines from the product suppliers. So the most successful standards will result from collaborative efforts between companies such as Axon and their customers."
Already Affymetrix, Inc., and Molecular Dynamics, a division of Amersham Biosciences, have formed the Genetic Analysis Technology Consortium (GATC). Its goal: to standardize array based genetic analysis, thereby paving the way for the more affordable and productive development of products for therapeutics, diagnostics, and disease management. It plans to do so by agreeing on a unified technology platform to design, process, read, and analyze DNA arrays. Researchers should benefit as GATC-compliant probe arrays, readers, reagents, and software and database architectures will eliminate the need for redundant equipment and software.
Few companies in the DNA chip business offer complete systems for working with DNA chips. Rather, most have focused on specific areas of technology and offer a limited range of products. Although this helps the companies to master their respective arts, it poses problems for researchers who need to assemble the pieces in their laboratories. Two notable exceptions are market pioneer Affymetrix and Agilent, which recently introduced a complete solution from arrays and labeling reagents to scanner and analysis software.
In addition, several companies have recently decided to work together to offer bundles of compatible microarray products. GeneMachines, a vendor of DNA synthesizers, microarray printers, and pens for spotting microarrays, provides an example. "We work very closely with several scanner manufacturers," says president and CEO Scott Hunicke-Smith. "We believe the market wants one-stop shopping, including the bioinformatics software needed to interpret microarray data."
The two basic types of DNA chips are differentiated by their method of production. One involves synthesizing oligonucleotides on a chip. The second spots presynthesized DNA (amplicons or oligonucleotides) onto a chip or glass slide.
Affymetrix originally developed oligonucleotide arrays by synthesizing oligonucleotides on a wafer or chip using a patented method for manufacturing integrated circuit chips borrowed from the semiconductor industry. The process involves using photolithography to add nucleic acid bases selectively to predetermined spots on the chip. This process enables the in situ (on a slide) creation of oligonucleotides up to 20 or so bases in length. This approach has one main disadvantage: high cost. Producing such a mask requires a substantial financial investment. Further, photolithographic masks cannot be altered to produce arrays with different oligos without incurring further cost.
Agilent has developed an alternative method. Exploiting its technological heritage, the company uses inkjets to deliver traditional phosphoramides and activaters to discrete locations on a modified glass support. By using traditional chemistries with high coupling efficiencies, Agilent can offer arrays of oligos 60 nucleotides in length by an in situ process.
With its series of GeneChip products, Affymetrix is one of several companies that cater to researchers who do not wish to master the art of making their own chips. Other firms offering DNA microarrays ready to use for popular applications include Clontech Laboratories and Azign Bioscience A/S (formerly Display Systems Biotech), whose discoverARRAY slides have over 2,400 expressed fragments of complementary DNA (cDNA). Mergen Ltd. also provides such arrays.
A second method of producing a DNA chip involves spotting DNA (again amplicons or oligonucleotides) onto a slide. Users can obtain the cDNA from libraries or produce it from the mRNA (messenger RNA) of a cell. Pat Brown of Stanford University pioneered this process. The Brown laboratory has gained a worldwide reputation for its promotion of DNA chips. Its website features a wealth of information on how to make and use DNA chips.
Producing a cDNA chip requires an intermediate step that Affymetrix's production method doesn't need: the use of reverse transcriptase polymerase to generate a library and the polymerase chain reaction (PCR) to convert cDNA into an amplicon. In recent years the polymerase chain reaction and reverse transcriptase have advanced from art to science, largely owing to the efforts of several companies to create more reliable ways of amplifying nucleic acids. Suppliers such as Amersham Biosciences, Invitrogen, Promega, QIAGEN, and Stratagene have developed kits and complete systems that have taken much of the trial and error out of amplifying DNA or RNA. "For individuals who don't want to make their own PCR products we offer ready-to-spot human PCRs," says Becky Mullinax, senior staff scientist at Stratagene. "We'll do the same for mouse PCRs in the middle of this year."
MWG-Biotech AG focuses on synthesizing oligos first and coupling them to the substrate surface afterward. "The main disadvantage of Affymetrix's on-chip synthesis is the limitation in the length of the oligos," says Donner. "We can purify the oligos and check them prior to spotting, which isn't possible with on-chip synthesis."
Different oligo makers provide the market with a choice among the lengths of oligos available for purchase. Some vendors regard the 20-mer length of Affymetrix's oligos as too limiting. "We use 50-mers," says Donner. Why? "It's a matter of production," he explains. "Our mass spectrometry can check oligos only up to 50-mers. And tests we have carried out suggest that 50-mers are highly specific, while oligos with more than 50 base pairs can lead to cross-hybridization."
QIAGEN Operon has a different take on the issue. "We have decided to use 70-mers and one oligo per gene," says Potgeter. "They are more selective. And scientists like to see the message coming from the tissue. The role of Operon is to support our customized chips with longer oligos." Agilent splits the difference. "We typically use 60-mer oligos rather than the shorter ones," says Amorese.
Whatever the selected length, it's clear that customers have increasingly opted for oligo arrays in recent months. "There's a move away from cDNAs to long oligos," says TIGR's Quakenbush. "I see an evolution toward these long oligos to ease sample handling. Complementary DNAs are hard to work with over a long period of time. Oligos are easy to maintain and reproduce. The challenge of using long oligos is that you rely on having a good working knowledge of what the genes are."
Millennium, which up to now has largely made and used its own cDNA arrays with nylon substrates, has recently started to take the oligo route. "We are substantially ramping up our use of Affymetrix microarrays," says Dionne. He cites three reasons for the change. "First," he says, "we think the Affymetrix platform has improved to the point at which it rivals our nylon platform. Second, the oligo base will have significant advantages in terms of giving us the ability to query certain parts of the genome that cDNAs can't query. And third, it gives us the opportunity to shift our resources downstream because we don't have to produce our own arrays."
In general, any scientific group must prepare or purchase a slide or chip, synthesize or obtain the DNA, apply the DNA samples to the chip, conduct a hybridization experiment with a sample of interest, scan the chip, and analyze the data. Several suppliers have entered the DNA chip market recently, providing the tools needed to produce chips as well as ready-to-use chips for popular applications.
DNA is attached to a solid substrate, such as a silicon/ceramic wafer, glass microscope slide, or a nylon membrane. Some of the early work with glass microscope slides was complicated by the fact that glass slides required pretreatment with such chemicals as polylysine to prepare the surface for DNA attachment. Those slides had poor lot-to-lot consistency. However, it did not take long for suppliers to develop better performing products. Corning, Inc., quickly introduced its CMT-GAPS aminosilanized slides. These offered lower background noise and better consistency. Corning has recently left the microarray business. However, other companies offering the latest in prepared slides for DNA chip production include Clontech, Genpak (a part of British company Genetix Group PLC), and Schleicher & Schuell.
Quakenbush at TIGR has successfully used super amine slides. "We are also getting away from harsh chemical treatments," he says. "I've been told that we make the best arrays that people have seen."
For genomic research, the entire genome of an organism is extracted from cells or tissue. Gene expression studies need either total RNA or mRNA for DNA synthesis. The purity of RNA is a critical factor in the hybridization step when labeling with fluorescent tags. Proteins or other contaminants can promote significant nonspecific binding to the slide's surface of fluorescent-labeled target DNA.
A few companies focus almost exclusively on providing tools for this segment of microarray manufacture. QIAGEN offers a broad range of reagents and kits for isolating and purifying DNA and RNA. "The better the preparation, the better the results," says Potgeter. "Your RNA has to be intact. Hence we have a program of RNA stabilization. One of our products is RNeasy Protect for tissues." In a recent collaboration with BD Biosciences, QIAGEN has developed a method of collecting blood and stabilizing it in a container. "We try to avoid any destruction of RNA — not just to get rid of shortened strands but also to avoid instability from RNA lying around for a long time," Potgeter continues.
Alternatively, scientists can buy oligonucleotides from companies such as MWG-Biotech, QIAGEN Operon, and Sigma Genosys. These suppliers provide custom oligonucleotide synthesis services. "We deliver catalog microarrays premanufactured," says MWG's Donner. "We also offer custom-made ones from lists of genes sent by customers; we then design the oligonucleotides and offer oligo sets based on our catalog arrays."
In mechanical spotting, a robotic liquid handling system picks up samples of DNA and deposits them onto the surface of a slide at predetermined locations. The pins used in this system, which can be split quills or solid rods, first come into contact with the DNA sample. Then they apply the sample to the slide by making contact with the slide surface. Washing the pins between applications ensures that any particular DNA sample is not cross-contaminated with another. Suppliers of robotic systems for microarray spotting include BioRobotics Ltd., Cartesian Technologies, GeneMachines, MiraiBio (Hitachi Genetic Systems), and Packard BioScience. This type of spotting system is very popular in academic laboratories owing to its ease of use, low cost, and versatility.
Cartesian Technologies, recently acquired by Genomic Solutions, sells a range of systems for spotting microarrays. "We offer a number of systems ranging from the small benchtop MicroSys, an 11-slide machine for entry level microarraying, up to the PixSys for 50 slides and the 100-slide ProSys," says Don Rose, Cartesian's vice president of research and development. "We're coming out with a new machine, the MegaSys, that will handle about 250 slides and will include a vision system for quality control and automated plate handling." Both systems can be configured with mechanical pins or inkjets.
Inkjet technology was adapted from the personal computer printer industry. A DNA sample is loaded into a tiny nozzle equipped with a piezoelectric device. The device expels a precise amount of DNA from the nozzle onto the slide surface. The nozzle is washed after each deposition to provide a clean nozzle ready for the next sample.
Agilent uses inkjet printing technology developed by its corporate parent, Hewlett-Packard, to manufacture its DNA microarrays. "One thing I find most intriguing about inkjets is the precise volume you can dispense," says Amorese. "It has a profound effect on arrays that every drop fired from the head is essentially the same volume." Agilent uses inkjet heads in two array methods. "For in situ arrays we synthesize oligos in place, using heads that have a continuous feed," Amorese says. "We can also deposit a presynthesized material from inkjets whose heads are filled with very small amounts of material." Other companies that provide inkjet technology include BioRobotics and Packard BioScience.
In multiplexing, each probe is labeled with a different fluor that can be simultaneously detected at different wavelengths with optical filters. An RNA sample is converted to labeled cDNA by reverse transcriptase PCR in the presence of fluorescently labeled nucleotide precursors. Genomic DNA is fluorescently labeled by nick translation or random primer techniques. To enable the direct comparison of two samples, scientists label them with different fluors, and then mix them together and hybridize them with a microarray. Agilent, Genpak and Roche Molecular Biochemicals, among other firms, also offer kits for labeling nucleic acid samples.
After hybridization, DNA chips are scanned using instruments specifically designed to detect fluorescent signals. Most manufacturers of imaging instruments use the same principle. A scanning fluorescence microscope illuminates each DNA feature or spot and measures the fluorescence of each fluor or dye separately. Scientists then use these measurements to determine the ratio or relative abundance of the sequence of each specific gene in the two mRNA or DNA samples.
"Right now there are two basic technologies for scanning," says Axon's Pickett. "One uses laser excitation and photomultiplier tube detection. The other uses a white-white system with filters for excitation and a CCD array for image collection." Axon's 4000 B laser scanner is a two-color system. "Its primary benefit is that it is a high performance instrument that is integrated with our GenePix software," says Pickett. "Our customers find it to be easy to use."
QIAGEN's new ultrasensitive SensiChip Reader uses cutting edge planar waveguide technology for microarray analysis. "By coupling the laser light through a diffractive grating onto the waveguiding film of our SensiChip microarrays, we can create an evanescent field whose excitation capacity for fluorescent dyes is limited to 300-400 nanometers," says Potgeter. "The CCD camera detects only the layer with the capture probes and their specific fluorescent targets. This dramatically improves the signal-to-background ratio." Other companies with expertise in such imaging detection include Agilent, GSI Lumonics, Genomic Solutions, and Molecular Dynamics.
Making sense of the large amounts of data collected from gene sequencing and gene expression experiments is no simple task. Scientists who need to manage and analyze the results from DNA microarray work spend many hours working with computers and specialized software to store and manage sequence data, design microarray formats, and analyze the data gathered from their studies. They can obtain help, though, via software solutions developed by BioDiscovery, GeneData AG, LION Bioscience AG, Scanalytics, Silicon Genetics, and Spotfire, among other vendors.
Public databases allow researchers to share the results of their work through the Internet. Researchers can query the large number now available to seek similarities and other relationships between different sets of data. One useful and accurate source of such databases is www.gene-chips.com. This website, maintained by Leming Shi of BASF Corporation, lists databases from the GATC, the National Center for Biotechnology Information (NCBI), the Stanford MicroArray Database, and others. It is also an excellent reference tool for any individual interested in working with microarrays.
Pharmaceutical companies embraced the use of DNA microarrays when they first became available because of their potential for improving the drug discovery process. Their scientists wanted to identify the genes involved in disease processes and to monitor responses to drug candidates. DNA microarrays offered them a new high throughput method for simultaneously evaluating large numbers of genes and monitoring physiological responses to potential drugs. Specifically, DNA microarrays can help researchers to find new drug targets — the molecules with which specific drugs interact — by identifying those genes whose expression levels are altered in a diseased state.
A more recent application for DNA chips occurs in the study of the natural DNA variations among individuals called single nucleotide polymorphisms (SNPs). A SNP is characterized by a single DNA base pair substitution at a specific location in a gene. For example, some individuals in a population may have the base "A" while others may have the base "C" at the same location. Taken together, many of these SNPs can be examined in an individual to develop a type of genetic fingerprint. Scientists can quickly examine the differences using a DNA microarray designed specifically for SNP analysis. This work has value because SNPs can provide information on an individual's predisposition to a given disease. They can even predict how a patient will respond to a particular class of drugs.
Rather than fixing the SNPs, PolyGenyx's proprietary OmniScan method fixes the genomes of multiple individuals to microarrays. "Through a single PCR reaction we can amplify a random reproducible set of the whole genome," Landers explains. Thus, the SNP detection step now involves hybridizing SNPs, rather than genomes, to the solid surface. This enables genomic DNA from over 10,000 individuals to be arrayed onto a single surface and genotyped simultaneously. This parallel processing approach is substantially more efficient than serial processing for larger population studies.
PolyGenyx is also developing proprietary technologies to enable simultaneous hybridization of multiple SNPs to a single microarrayed surface that will improve efficiency even further. In Landers's view, the method has potential uses in DNA fingerprinting. It could handle the huge backlog at the FBI and other crime labs," he says. "We've also had people ask us about the method's use in foods. It could answer such questions as 'Where does your caviar really come from?' and 'Do your coffee beans come from Colombia?'"
Dionne of Millennium sums up the promise of DNA microarrays and chips. "We think that the real future of microarrays will involve utilizing data better," he says. "That's clearly in the type of experiments done and the analysis and associating not only gene expression but also pathway expression types of data. That's where a lot of our focus is going to be."
Peter Gwynne is a freelance science writer based on Cape Cod, Massachusetts, U.S.A. Gary Heebner is a marketing consultant serving the scientific industry, based in Foristell, Missouri, U.S.A.
This article was published
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in the 4 January issue of Science