Array of Options

Instrumentation for Microarray Production and Analysis - Part 1 Instrumentation for Microarray Production and Analysis - Part 2 Nanogen's NanoChip™ Cartridge Today's molecular biology era can be defined by the dictum, "So many genes, so little time," and technologies for gathering genetic information are gaining speed. DNA microarrays are one of the most promising answers to that cry. DNA microarrays are glass microslides or nylon membranes containing DNA samples (genomic DNA, cDNA, or oligonucleotides) in an ordered two-dimensional matrix. DNA microarrays can be used to analyze gene expression and genomic clones or to detect single nucleotide polymorphisms (SNPs).1,2 The DNA used to create a microarray is often from a group of related genes such as those expressed in a particular tissue, during a certain developmental stage, in certain pathways, or after treatment with drugs or other agents. Expression of that group of genes is quantified by measuring the hybridization of fluorescently labeled RNA or DNA to the microarray-linked DNA sequences. By profiling gene expression, transcriptional changes can be monitored through organ and tissue development, microbiological infection, and tumor formation. Also known as biochips, DNA microarrays can be created by linking monomeric nucleotides on the glass surface to make oligonucleotides. Another methodology, popular for making arrays of PCR products and organismal genes, uses robotic instruments to spot thousands of DNA samples onto a surface. This high-throughput approach increases reproducibility and production. Making the arrays entails transferring 1-2 nl of DNA sample from 96/384/1,536-well microplates to a 100-200 µm spot on the glass microslide. This is accomplished through single spotting with solid pins or multiple spotting with "split" pins. Output is determined by the number of pins, input microplates, and output microslides. Microarray readers-- surface fluorometers--are also part of this equation. Since microarrays are used in university research, small and large biopharmaceutical companies, and large-scale clinical trial investigations, there are a variety of instruments and integrated systems to meet these diverse needs. This treatment will focus on tools for the needs of academic researchers, possibly the fastest-growing segment of microarraying scientists. Packard Instrument Co.'s BioChip Arrayer Helping Hands For arraying large DNA libraries, automatic colony pickers that can transfer genetic material from growth plates to multiwell microplates can reduce the workload. With the capacity to pick isolated colonies from 72 agar plates and output 24,000 colonies per day in 72 microplates, the Gel-2-Well™ from GeneMachines™ of San Carlos, Calif., is an able middle-range system. The BioPick automated colony picker is a robust desktop instrument from BioRobotics of Boston. The system is now available with an accessory for extracting a plug of phage from agar. The Q Bot, Q Pix, and Q Pix Stacker systems from Genetix of Christchurch, United Kingdom, use a patented 96-pin picking head and pick in excess of 4,000 colonies per hour from petri dishes or trays into standard microplates (96 or 384 well) or deep well blocks. A high-performance industrial-strength robot made completely in the United States, the AutoGenesys by AutoGen Inc. of Framingham, Mass., can handle up to 80 microplates (96 or 384 well) at 3,000 colonies per hour; it can run unattended for up to 14 hours using 384-well microplates. Another potentially troublesome step in obtaining high-throughput pace within the biochip universe is carrying out PCR. To perform PCR on four microplates at once, MJ Research Inc. of Waltham, Mass., provides the PTC-225 DNA Engine Tetrad, a Peltier-based thermal cycler that can be modularly expanded to use eight dual-microplate Alpha modules for 96- or 384-well plates. MWG-Biotech Inc. of High Point, N.C., offers the Primus Multiblock Legal PCR System, which allows users to add up to seven four-microplate modules and the RoboAmp® 4200, a high-end robot that can prepare one PCR microplate while it cycles another. Hybaid of Franklin, Mass., offers the MultiBlock System with a central computer that controls up to 30 individual cycling blocks. Blocks are available to fit 96- or 384-well microplates for high-throughput applications. Several systems for hybridizing probes to microarrays are available. TeleChem International Inc. of Sunnyvale, Calif., offers the ArrayIt™ hybridization cassette, an individual chamber for microslide- spotted arrays. GeneMachines offers the HybChamber™, a dual-biochip hybridization chamber that can be interlocked and stacked in water baths. Both use a few microliters of oligo solution. For larger incubation volumes is the Atlas™ Glass Hybridization Chamber from CLONTECH of Palo Alto, Calif., a capped vessel that allows hybridization in ~1.8 ml. Facilities with continuous sample flow will shoot for the GeneTAC™ Hybridization Station from Genomic Solutions of Ann Arbor, Mich., the only available multichamber automated system. This stand-alone system has an internal computer and software that allows precise temperature, volume, and timing controls. Six dual-biochip chambers can be filled with up to 12 microarray slides. To help incorporate robotics into university-based facilities, companies such as ROBOCON of Vienna, Austria, offer piecemeal solutions for a number of steps: multiplate incubators, warmers, and shakers; a transfer and reagent addition station; and microplate pick-and-place devices for multiplate thermal cyclers or ROBOCON's ROBOLAB microplate centrifuge. Vysis' GenoSensor Reader System If the Suite Fits Several powerful systems provide specialized arrays plus all the instrumentation needed to read and analyze them. This treatment focuses on individual user- oriented suites rather than company-level solutions such as MTAP (Microarray Technology Access Programme) from Molecular Dynamics of Sunnyvale, Calif. GeneMachines' colony-picking Gel-2-Well™ and OmniGrid™ arrayer are stand-alone instruments that can be integrated with their high-throughput DNA purification, oligo synthesis, DNA shearing, and bacterial growth modules. Seamless integration of available instruments and data output from the OmniGrid compensate for the lack of a scanner.3 Another integrated system is Genomic Solutions' GeneTAC biochip instrument. A multiple-function Flexys® Robotic Workstation fulfills many high-throughput needs for the microarray lab: It can grid DNA or cDNA libraries onto nylon membranes or spot them on microchip glass slides to create microarrays; using a CCD camera, it acts as a colony-picking robot with 99 percent accuracy and the flexibility to fully rearrange libraries, arraying and rearraying its contents on 96- or 384-well plates. Genomic Solutions offers services to make carefully controlled microarrays from DNA libraries, prepare fluorescent probes from mRNA, and even hybridize them. SeqWright of Houston, Agilent Technologies of Palo Alto, Calif., and TeleChem also offer customized arraying services. Affymetrix of Santa Clara, Calif., provides high-volume production methods that can support the diagnostics or drug development industries. Affymetrix offers GeneChip® technology, which uses glass microarrays manufactured by a proprietary process that combines solid-phase chemistry and photolithography to build probes in situ. The glass wafers are packaged in plastic cartridges in which hybridization is carried out. Several hardware components form the GeneChip suite. The GeneChip Fluidics Station 400 introduces the sample into the probe array cartridge. The Hybridization Oven 640 processes up to 64 cartridges. Agilent Technologies designed its GeneArray scanner (monochrome; 20 µm resolution) to be used exclusively with Affymetrix microarrays, and the scanner is distributed by Affymetrix for integration into the GeneChip suite. Affymetrix also offers a series of software solutions for data collection, conversion to GATC™ (Genetic Analysis Technology Consortium) database format, data mining, and a multiuser LIMS system for power-hungry environments. Hitting the Spot Arrayers do the dirty work of dipping into microplate wells containing DNA and precisely spotting nl-size drops onto a surface grid. A popular arrayer meeting the needs of university-based facilities is GeneMachines' OmniGrid™. The OmniGrid uses four 96- or 384-well plates to create 100 slides with 64,000 spots (50 µm spot size) in 17 hours. An upgrade to 36 input plates will be available soon, and GeneMachines has recently improved the software capabilities to include customizable wash sequences, printing and bar code integration, and security access options. In addition to the GeneChip-specific suite of products, Affymetrix offers the Affymetrix 417 arrayer. This instrument uses Pin-and-Ring™ technology to acquire fluids from microplates and transfer precise amounts to the microarray grid.4 The arrangement allows arraying with liquids of very different viscosities (oligos, peptides, antibodies) onto many substrates (uncoated, derivatized glass slides, cover slips, porous membranes, gels, plastics). The unit handles three 96- or 384-well plates and 42 slides with four pins (125 µm spot). Whereas production environments tend to mass-produce a narrow range of arrays, academic facilities must be able to handle many types of array production. BioRobotics offers the MicroGrid TAS (Total Array System) that grids up to 100,000 features onto glass slides or membranes; the improved MicroGrid II (120,000 features/array) sports automatic data handling via bar coding and complete environmental controls. The production-oriented MicroGrid Pro grids 64 features per second. With up to 64 MicroSpot 2500 pins/head, the MicroGrid arrayers use 24 source plates (96, 384, or 1,536 wells) and output over 100 slides with spot sizes of 80-140µm. Data output is compatible with most software packages. Cartesian Technologies of Irvine, Calif., has developed arrayers around two types of printing technologies. The PA series uses PinArray™ technology with up to 48 noncontact Stealth™ pins (TeleChem) to handle up to 50 plates and 100 slides (75-200 µm spots). The NQ series uses nQUAD™ (nanoliter Quantitative Aspirate and Dispense) technology, similar to inkjet printing, for up to 100 output slides. The production-level PegaSys PA raises the bar to 100 source plates and 1,000 slides, and helpful plate autoloaders are available for other models. TeleChem offers the ChipMaker™ Models 2 (32 pins, 100-150µm spots) and 3 (48 pins, 75-100 µm spots), which can produce 50 biochips with 62,000 spots in less than 24 hours. Other entries in this field include the Q Array from Genetix, which features humidity and environmental control, 1 µm resolution, and 84-slide capacity. The new SPBIO™ Microarray Spotting Station from Hitachi Genetic Systems of Alameda, Calif., features eight or 32 solid pin heads for 96- or 384-well plates, respectively. The instrument has a 14-microplate input with optional stacker and fast output of 15,000 spots per hour. The BioChip Arrayer™ from Packard Instrument Company of Meriden, Conn., uses PiezoTip™ piezoelectric technology for noncontact transfer. The BioChip Arrayer has four tip heads, handles 96-, 384-, and 1,536-well microplates, and outputs 25 slides with 200µm spots. Noncontact transfer allows access to any microarray surface and produces low variability between spots. Somewhat outside the boundaries of this group, Nanogen of San Diego has developed a machine that uses microelectronics to electrochemically direct DNA or other biological molecules to specific locations on biochips. Its current cartridge-based system addresses DNA to 100 electrodes on the microcircuit. An advanced prototype, developed in a research program with Aventis, has 10,000 independently programmable electrodes. An inherent advantage of this futuristic system is reduced hybridization times, a foreseeable need for diagnostic labs. Touched by Light A host of efficient microarray readers have capitalized on existing confocal and imaging technologies and are quickly incorporating robotic platforms. An overall single-microslide capacity favorite is the GenePix 4000 A DNA scanner from Axon Instruments Inc. of Foster City, Calif., which offers a great deal of functionality and integration. The GenePix simultaneously measures at two wavelengths (532 and 635 nm) and can collect fluorescent ratios in real time; it has high sensitivity, accurate alignment, and can scan in less than five minutes at 10 µm resolution. The unit reads bar codes automatically and has fully integrated acquisition-analysis software (GenePix Pro 3.0). Alternatively, Affymetrix offers a dual-fluorescence Affymetrix 418 scanner that detects Cy3 and Cy5. This scanner has rapid scanning technology (Flying Objective™), with a proprietary high-numerical-aperture lens and confocal, epifluorescence geometry. The ChipReader™ from Virtek of Ontario, Canada, is a multicolor, small, and fast scanner that can read a 20 x 20 mm surface in less than a minute. For high-volume environments, a powerful multisample scanner is part of Genomic Solutions' GeneTAC Biochip solution. The GeneTAC 2000 BioChip Analyzer can scan up to 24 microarrays using a carousel, and with a filter wheel, allows detection of up to 10 fluorescent labels. An upscale solution is the GeneTAC LS IV Biochip Analyzer, allowing high-throughput, four color-based analysis (three internal lasers and one external). The AlphaArray™ from Alpha Innotech of San Leandro, Calif., can analyze 12 conventional slides, one 3 x 5 in. slide, or two 96-, 384-, or 1,536-well plates. For facilities with many specialized needs, GSI Lumonics Inc. of Billerica, Mass., has an incremental line of scanners including the ScanArray® Lite (two internal lasers), the ScanArray 4000 (three colors/ three lasers, seven emission filters), and the high-end ScanArray 5000 (four colors/four lasers, 10 emission filters). All have 5 µm-resolution and come with QuantArray® analysis software. All of the scanners in this series are upgradable and available with bar code reading capabilities. A microarray autoloader system can also be added to the ScanArray 4000 and 5000 systems. Future microarray readers will go beyond scanning and profit from imaging technology that uses CCD cameras. Scanners capable of total image measurement include Alpha Innotech's AlphaArray™, measuring up to eight fluorescence dyes, Hitachi Genetic Systems' CRBIO™ featuring interchangeable filters and flexible scanning time, and the GenoSensor™ reader from Vysis of Downers Grove, Ill., which scans Vysis microarrays (0.5 cm2) in less than a minute and provides up to six fluorescent labeling colors and reader software that identifies gene targets and presents results for data analysis. Two performance features to consider when buying a scanner are the number of lasers (colors) and the number of microslides per unit. Axon's GenePix does a great job of handling one slide at a time, but for a large facility, systems such as Genomic Solutions' GeneTAC Array Analyzer will carry the day. The more scientists involved, the more likely capacity and fluorophore flexibility will become an issue. Software Savvy Software tools in the DNA microarray world form by themselves a large array. Some are configured to operate within specific machines; others are tailored to exploit array-specific systems such as Affymetrix's GeneChip, GeneFilters® from Research Genetics of Huntsville, Ala., and GEM™ microarrays from Incyte Genomics of Palo Alto, Calif. A third group involves several externally compatible packages for the general user. BioDiscovery of Los Angeles offers several software solutions for all levels of user needs. ImaGene™ 3.0 Image Quantification Software offers many features for gene expression measurements with fast analysis speed (10,000 spots can be processed in less than two minutes); it can be customized with templates tailored to the products from various manufacturers. GeneSight™ is its data mining software, which produces 3-D scatter plots, hierarchical and neural network clustering analysis (to pool groups of similarly sensitive genes), and time series plots (to compare early- and late-expressing genes). Its newly developed AutoGene™ is the first completely automatic microarray analysis software; it uses vision algorithms to remove image contamination and correct for spot irregularities.5 ArrayVision™ from Imaging Research Inc. of Ontario, Canada, is array quantification software compatible with all popular fluorescence and phosphor plate scanners. It offers array-analysis automation, extensive quality metrics, artifact detection, and Flexible Interpolating Targeting (FIT), which automatically corrects warps. Spotfire Array Explorer™ (SAE) from Spotfire Inc. of Cambridge, Mass., interfaces with GATC databases, reads Incyte LifeArray® files, imports data from Genomic Solutions' GeneTAC Integrator software, and is integrated with Molecular Dynamics' Experiment Manager. Spotfire offers SAE as an integrated Scientific Component in its Spotfire.net decision analytics core offering. Spotfire.net has a Web-based portal that allows data access, interactive data analysis, and worldwide collaborations among teams and departments. QuantArray™ from GSI Lumonics is a Windows® NT package that offers automated quantification and analysis of up to four-color microarray images, with spot finding and fluorescence crosstalk correction that is completely automatic when used with ScanArray instruments. For die-hard Macintosh® users, there is the MicroArray suite, constructed as a modular extension of the IPLab image analysis program from Scanalytics of Fairfax, Va. Demos of some microarray software are available from company Web sites or at scientific conferences for those who would like to try the often complex programs before committing. The GATC standard will eventually allow companies to create more automatic, integral solutions and add even more statistical tools to satisfy the needs of clinical trial users. An effort in this direction is CHIPSpace™ Chip Information Management Environment from Hitachi Genetic Systems, which will manage software needs all the way through arraying, hybridizing, measuring, and analyzing spots. In a high-throughput environment, bottlenecks have a multiplicative--rather than additive--effect on production time. Plainly, choosing a system that cannot be easily upgraded can decrease the maximal number of users substantially. In a 50-100 user facility, that's a lot of unhappy colleagues. Also, increasing the overall output may mean replacing several very expensive components at once. A balance between needs and means is essential in designing an operationally sound setting, and according to Laura Reid, director of the genomics core at the University of North Carolina at Chapel Hill, "To prepare microarrays, the entire lab has to get into the high-throughput mode." Jorge Cortese can be contacted at jorge_cortese@mindspring.com. References 1. M. Schena et al., "Quantitative monitoring of gene expression patterns with a complementary DNA microarray," Science, 270:467-70, 1995. 2. S. Solinas-Toldo et al., "Matrix-based comparative genomic hybridization: biochips to screen for genomic imbalances," Genes, Chromosomes & Cancer, 20:339, 1997. 3. B. Sinclair, "Suite dreams," The Scientist, 14[2]:22, Jan. 24, 2000. 4. B. Sinclair, "Everything's great when it sits on a chip," The Scientist, 13[11]:18, May 24, 1999. 5. A. Constans, "That's the spot!" The Scientist, 15[5]:24, March 6, 2000.

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