The Array of Today

Human Arrays Human Arrays (continued) A 20th century scientist may have spent an entire career studying the function of a single protein, never imagining that one day it would be possible to study every human gene at once. This capability comes in the form of a microarray, a surface collection of immobilized genes that can be simultaneously examined with specialized equipment.1 Many current applications of arrays, also known as biochips, can be used in functional genomics as scientists seek characteristic patterns of gene expression in different physiopathological states or tissues. A step beyond this, genomic linkage analysis associates genetic markers with disease phenotypes. Arrays also can support massive high-throughput genomic screening, assessing single-base genome changes (single nucleotide polymorphisms, or SNPs) that underlie individual variations in drug response. With many potential applications and the approaching genome explosion, a host of commercial arrays and array tools have suddenly appeared. This article examines the available array systems and peripheral technologies. The idea of having arrays of thousands of genes is very appealing to genomic scientists, and creating such librarylike arrays has been a prime goal of many biotechnology companies. These arrays, produced through a variety of technologies, consist of either complementary DNA (cDNA) clones or oligonucleotides. Incyte Genomics Inc. of Palo Alto, Calif., creates high-density microarrays by depositing and binding 500-5,000 bp cDNAs to a glass surface. The Human Genome GEM™ microarrays have between 8,616 and 9,604 unique genes, many with known function. Incyte also offers nonhuman microarrays as well as individual clones or clone sets from the GEM microarray collection. The company plans to release additional human, mouse, and rat microarrays before the end of 2000. Incyte also has a new line of nylon membrane LifeGrid™ arrays, with a broad selection of sequence-verified clones from human, mouse, or Arabidopsis thaliana. Another company producing cDNA arrays is NEN Life Science Products Inc. of Boston. The company collaborates with AlphaGene Inc. of Woburn, Mass., to offer the MICROMAX™ human cDNA I array, glass slides containing 2,400 known genes with 2,200 bp average length. CLONTECH of Palo Alto, Calif., creates glass arrays with single-stranded DNA (80-nucleotide oligos) and nylon arrays with 200-600 bp PCR products. Each arrayed element is optimized to avoid homology with other genes in the array. CLONTECH prints its Atlas™ glass arrays on DNA-Ready™ Type II slides using a proprietary chemistry that facilitates DNA binding. Customized arrays on nylon membranes can be prepared with the company's PCR-Select™ cDNA subtraction method, which creates arrays with cDNA from differentially expressed genes. Affymetrix of Santa Clara, Calif., synthesizes oligonucleotides directly on the chip with computer-generated photolithographic masks. These masks define exposure sites on the chip's surface, where a light-directed synthesis process constructs the oligonucleotides combinatorially. Many arrays are synthesized simultaneously on a glass wafer and then packaged in injection-molded cartridges that serve as hybridization chambers (GeneChip® arrays). Affymetrix has developed a number of premade GeneChip arrays, from an Escherichia coli array with 4,200 known open reading frames (ORFs) to the Human Genome U95 Set containing probes for 12,000 full-length genes and 50,000 expressed sequence tags. Display Systems Biotech of Vista, Calif., uses restriction fragment differential display PCR (RFDD-PCR) technology to produce its discoveryARRAY™ and tissueprintARRAY™ Gene Display Slides, which include genes differentially up-regulated in one or more of eight human tissues. BD PharMingen of San Jose, Calif., a division of BD Biosciences, offers the BD RiboScreen™ Human 1 Membrane for screening 289 different human genes. Mergen Ltd. of San Leandro, Calif., synthesizes oligos off-chip, then spots and covalently links them to slides. Off-chip synthesis is more flexible and economical than on-chip chemistry. Mergen offers the ExpressChip™ DNA microarray system, a kit with two identical glass slides containing approximately 1,100 different genes, each selected for its relevance to cell growth or disease. The kit also includes a hybridization chamber and reagents for hybridization and detection of the fluorescent signal. There is an ExpressChip selection for human, mouse, and rat genes. Mergen utilizes a unique algorithm to design a single 30-nucleotide oligo based on sequences near the 3' end of each gene, with controlled melting temperatures and G/C content. Research Genetics of Huntsville, Ala., has focused on broad nylon membrane-based arrays (GeneFilters®). The Human Releases I-VII each consist of a single membrane spotted with cDNAs (approximately 1,000 bases from the 3' end). The DNA has been denatured and UV-crosslinked to the positively charged membrane. The company plans to release a new GeneFilters microarray every 90 days until it reaches its goal of 40,000 genes. GeneFilters users can strip and reuse the product; with careful use, up to five probings are possible. Large collections of human genes with known function and prostate-, ovary-, or breast-specific genes are available, as well as yeast ORFs and an ongoing rat and mouse release series. Vysis uses chromium-coated glass surfaces for assessing gene copy number changes. Focus Your Energy For researchers using microarrays to study genes involved in complex cascades of signal transduction events (such as apoptosis and cancer), various focused array systems have arrived. Sigma Genosys Inc. of The Woodlands, Texas, offers the Panorama™ collection of genes, a comprehensive macroarray of human apoptosis- and cytokine-related genes; standard radiolabeling hybridization techniques can analyze these genes. Other Panorama arrays cover bacterial genomes. The company also offers PCR primer pairs designed to amplify protein coding regions from genes on the filters. R&D Systems of Minneapolis also offers focused arrays on nylon membranes, the Human Apoptosis Expression Array and the Human Cytokine Expression Array. These arrays contain PCR products from completely sequenced cDNA clones and include positive and negative control genes. NEN Life Science Products has also developed a focused array system based on its MICROMAX glass arrays, covering signal transduction and cancer-related genes. Selections of stock arrays from apoptotic and cancer genes are also available as OpArrays®, the highly optimized oligo arrays produced by Operon Technologies Inc. of Alameda, Calif. Because OpArrays are sequence- optimized and their hybridization temperatures are normalized, specificity increases without sacrificing sensitivity. Affymetrix also offers focused arrays such as the GeneChip Human Cancer G110 Array and the Rat Neurobiology U34 Array. Moving from the known to the unknown, some companies manufacture arrays that contain possibly novel sequences. Stratagene of La Jolla, Calif., has introduced the GeneConnection™ discovery cDNA microarrays, containing 4,000 unique PCR products from the company's clone collection, many of which lack significant nucleotide homology to public databases. Stratagene also offers the individual clones for further in vitro analysis and provides the GeneConnection Virtual lab, in which clones can be queried through protein and nucleotide sequences. OriGene Technologies Inc. of Rockville, Md., has introduced the SmartArray™ gene chip series. Each microarray in the series contains a preselected group of genes in the form of PCR fragments (400-700 bp). These selected genes are functionally related and are involved in the same regulatory pathway; the focused chips allow users to identify candidate genes with products that interact and/or act in concert to control cellular functions. Each PCR fragment was selected to have more than 85 percent homology with its mouse counterpart but not to hybridize with other homologous members of the same gene family. The SmartArray chips are prepared on standard microscope slides with a nitrocellulose membrane overlay. Users can employ multiple detection methods, including radioisotopic (33P), chemiluminescent, or fluorescent detection. When radioisotopic probes are used, the chips can be stripped and reused up to three times. Taking the focused array idea to the extreme with custom- and program-ordered arrays, SuperArray Inc. of Bethesda, Md., is developing many pathway-specific arrays for radioactive and nonradioactive detection. SuperArray's GEArrays™ are obtained from human and mouse genes. GEArrays can also be tailored (Human and Mouse Choice GEArrays™) with genes selected from SuperArray's Gene Bank. Vysis Inc. of Downers Grove, Ill., offers the GenoSensor system, which combines instrumentation, software, and arrays to determine gene copy number changes. Vysis' arrays are based on BAC/P1-type clones; genes are spotted onto a unique chromium-coated glass surface, which limits the autofluorescence of the glass. Vysis currently offers the AmpliOnc™ I Microarray Kit, which contains more than 50 gene targets for use in gene amplification studies; the company is developing a second kit for studying tumor suppressors. Additionally, Vysis plans to develop arrays for pre- and postnatal screening. Get with the Program Several companies offer services that cover all stages of microarray production and analysis. Mergen helps with oligonucleotide synthesis, spotting, hybridization, scanning, and data analysis. Operon Technologies can design and prepare custom, optimized 70-mer oligos (OPTs®), sold either as "array-ready" products or printed as arrays. Radius Biosciences Inc. of Medfield, Mass., specializes in custom arrays of DNA, RNA, oligos, PNA (peptide nucleic acid), proteins, or antibodies. Genomic Solutions Inc. of Ann Arbor, Mich., offers the GeneMAP Microarray Kit and a full line of custom services. SeqWright of Houston, Texas, will grid DNA onto nylon membranes (up to 57,600 clones per filter). MWG-Biotech Inc. of High Point, N.C., offers an array design service for individual requirements. Services include spotting of special HPSF® (highly purified, salt-free) grade oligonucleotides, optimized for specificity, as well as purified PCR products. Affymetrix licenses MWG to manufacture, use, and sell oligos and cDNA chips. Incyte Genomics, NEN Life Science Products, Mergen, and Genomic Solutions offer different degrees of array processing capability for hire. For example, NEN will scan its MICROMAX arrays, returning a CD with data and graphic representations to the user. Sophisticated services from TeleChem International Inc. of Sunnyvale, Calif., and CLONTECH approach full-strength microarray programs. TeleChem will manufacture custom microarrays on most surfaces in its state-of-the-art cleanroom. For its FlexChips™ service, customers send samples (nucleic acids, proteins, and small molecules) in a 384-well microplate, and TeleChem prints them on an optically flat, superaldehyde microarray substrate. TeleChem's e-Chips™ service allows customers to mine databases and send DNA, RNA, or PNA sequences of interest to TeleChem by E-mail for custom arrays. CLONTECH offers custom Atlas services that range from production of custom-designed nylon or glass arrays to hybridization and analysis. Display Systems Biotech is also creating a programlike environment for its customers. The PROFILE to ARRAY™ program provides a powerful approach for rapidly isolating and arraying expressed sequences from any organism to create specialized cDNA microarrays. This program also offers individualized clustering analysis of these custom-designed microarrays to compare multiple samples. Some programs surround the user with all elements of microarray technology. The differences between services is subtle, mostly at the level of sample turnout. Affymetrix runs the oldest program in the microarray world, with a large base of users worldwide. An integrated instrumentation system and three software systems support Affymetrix's GeneChip arrays.1 Agilent Technologies Inc. of Palo Alto, Calif., has a DNA Microarray Technology Access Program (TAP) that provides users with early and preferential access to cutting-edge microarray technologies. Agilent will produce custom oligonucleotide and cDNA arrays on glass slides, provide the TAP customers with an advanced, highly automated DNA microarray reader, and deliver expert support, consulting, and optimized protocols. Agilent has teamed up with Rosetta Inpharmatics Inc. of Kirkland, Wash., to offer an enterprisewide system for the analysis of gene expression data. Amersham Pharmacia Biotech of Uppsala, Sweden, provides all elements to fabricate arrays with instruments, reagents, and software. Systems can be tailored to both low- and high-throughput users, and technical support and training are an intrinsic part of the program deliverables. Hyseq uses microarrays for DNA sequencing. All Things Arrayed Companies are frequently modifying array technologies. For instance, Hyseq Inc. of Sunnyvale, Calif., has applied the array concept to sequencing by creating the HyChip™, a universal sequencing chip. HyChip uses an array of complete DNA probes plus a target-specific cocktail of labeled probes to sequence DNA from any source without previous sequence knowledge. Gene Logic Inc. of Gaithersburg, Md., is using the perpendicular dimension of arrays to facilitate probe-target interactions. Its Flow-Thru Chip™ probe array contains microscopic channels with probes attached to the walls; sample molecules flow through the channels and bind to the probes. The large surface area and uniform microstructure of the channel afford greater analyte sensitivity and faster reaction speed when compared with similar analyses performed on 2-D probe arrays. To push the detection envelope, SEQUENOM of San Diego offers the SpectroCHIP™, a two-dimensional array (384 spots) compatible with MALDI-TOF (matrix-assisted laser desorption/ionization time-of-flight) mass spectrometry. The SpectroCHIP is a component of the MassARRAY™ system, which allows high-throughput genomic analysis of SNPs (up to 20,000 results per day). A problem with many array production technologies is that array design is fixed, supported by costly photolithographic masks. Rosetta Inpharmatics has created a highly flexible microarray platform in which modifying arrays is as easy as changing a color in inkjet printing. Thus, product users can change array design at will and inexpensively, allowing iterative study of experimental results. Rosetta places up to 50,000 genes on a 3 x 3 in. glass wafer. The wafer is then sliced into two 1 x 3 in. microscopic slides with up to 25,000 genes per slide. The technology (FlexJet™ DNA microarrays) is available through in-house collaborations with Rosetta and is commercially available through Rosetta's strategic partner, Agilent Technologies. Another fast printlike arraying process is the DNA FlexChip™ technology from ProtoGene Laboratories Inc. of Menlo Park, Calif. ProtoGene manufactures custom DNA chips based on in situ synthesis of oligos. The company uses its patented surface tension technology combined with standard phosphoramidite chemistry to synthesize oligonucleotides directly on the chip. The technology's flexibility allows custom chip designs to be turned around in a matter of days, permitting the researcher to perform real-time iterative biological experiments. Maskless technologies such as these promise to change the array field; for instance, Texas Instruments Inc. of Plano, Texas, has produced a new Maskless Array Synthesizer based on computer-linked micromirror arrays.3 Companies are exploring immobilization technologies to address problems with probe-array linkage technology. Mosaic Technologies Inc. of Waltham, Mass., SurModics Inc. of Eden Prairie, Minn., and Prolinx® Inc. of Bothell, Wash., offer novel small-molecule linkers. Prolinx's Versalinx™ System, for example, is based on small synthetic molecules and is suitable for arrays of all varieties. Mosaic Technologies is developing a robust attachment technology to allow coupling of Acrydite™ oligonucleotides to glass slides with a proprietary polymeric coating. INTERACTIVA Biotechnology of Ulm, Germany, is creating arrays that combine photolithographic techniques with streptavidin-based immobilization. Ciphergen Biosystems Inc. of Fremont, Calif., has made first claims to the protein array territory with its ProteinChip® System. The system consists of a time-of-flight mass spectrometry reader, software, and ProteinChip arrays. The eight-spot arrays are available in a wide range of surface chemistries, aiming to capture, purify, and characterize chip-bound proteins. The arrays are readily fit for separation, detection, and analysis of proteins at the femtomole level by SELDI (surface-enhanced laser desorption/ionization). Enrique Dalmasso, senior scientist at Ciphergen, emphasizes that its ProteinChip technology will contribute to proteomic and protein functional studies by monitoring protein biomarkers during disease development and drug treatment, finding ligands for orphan receptors, and characterizing novel signal transduction pathways. Another protein array player is Phylos of Lexington, Mass. It is developing the first high-throughput protein biochip (PROfile™ Chip) that will allow scientists to scrutinize hundreds and ultimately thousands of distinct proteins contained in a complex biological sample such as blood or human tissue. Each feature on this addressable array comprises a "capture protein," engineered for high affinity and specificity using Phylos' proprietary protein evolution technology (PROfusion™). When a biological sample passes over the chip, specific capture proteins will bind only to corresponding target proteins in the sample. In this dreamlike field of arrays, users are in the driver's seat: If they want to travel the array highway, someone will build it. Jorge D. Cortese (jorge_cortese@mindspring.com) is a freelance writer in Durham, N.C. References 1. J.D. Cortese, "Array of options," The Scientist, 14[11]:26-9, May 29, 2000. 2. M.D. Brush, "The fast lane," The Scientist, 14[9]:21, May 1, 2000. 3. S. Singh-Gasson et al., "Maskless fabrication of light-directed oligonucleotide arrays using a micromirror array," Nature Biotechnology, 17:974-8, Oct. 17, 1999.

The Array of Today

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