Suite Dreams

Most researchers would agree that manually performing a hundred or so plasmid preps after a low-efficiency cloning stifles the spirit of exploration that attracted them to science. Minipreps don't end with merely being tedious, repetitive, and time consuming--frequent exposure to hazardous chemicals adds to the misery. However, minipreps are a necessary evil in research, and the number of sequencing templates to prepare never seems to dwindle. For example, a small lab running four sequencing gels a day with a 36-well comb can accommodate 144 samples a day; a larger operation running just one 96-capillary machine around the clock can analyze 1,000 templates daily. Preparing templates to meet this demand is challenging. DNA purifications are not the only tedious and repetitive tasks associated with today's genomic world. The small lab in the above example, at 450 bases per read, will generate about 65 kb of raw sequencing data per day; the larger operation, at 550 bases per read, will generate close to 650 kb of raw sequence. Assuming this is random (shotgun) material at 10-fold redundancy, the small lab can sequence one ~6 kb insert from a plasmid library each day; the larger operation might sequence four average BAC or 15 cosmid inserts. For high-throughput sequencing, each of these chunks of DNA must be subcloned in a small vector adjacent to a universal primer site. So once the primary library has been constructed, even a small lab needs to complete an entire shotgun subcloning and screening every day to feed the machine. The situation is even more intense in the larger operation and takes on nightmarish proportions in the high-throughput facilities that have several (or several dozen) big sequencing engines. Fortunately, rather than forcing too many graduate students into law school, a variety of liquid handlers and robotic workstations (reviewed in LabConsumer, Feb. 15, 1999)1 have been created to do repetitive work. The traditional approach to laboratory robotics and automated liquid handlers is to create a machine that is flexible enough to accommodate a range of different protocols. A typical scenario is for a lab to purchase a liquid handler and continue using the same protocols they used for manual techniques. The liquid handler compresses 96 (for 1.7 ml tubes and a single-tip pipette) or 12 (for a multititer dish and an eight-head multipipette) pipetting operations into one. However, the operator still usually needs to perform many of the manipulations: mixing the reagents in the plate, centrifuging or vacuum filtering the reactions, and so forth. Moving up to a robotic workstation--if you have sufficient bench space--can eliminate many operator steps, as it can move plates from here to there using some form of gripper tool. While most manufacturers can help to some degree with protocols, labs frequently continue to use their existing methods and often end up with significant start-up and troubleshooting problems. GeneMachines (San Carlos, Calif.) has taken a different approach to providing automated solutions for moderate-to-high-throughput labs, moving tried-and-tested instruments and protocols out of the specialist genomic laboratories and into the marketplace. GeneSuite is GeneMachines' collection of DNA tools for genomic research. "Our real goal here is to strive for a suite of instruments and to provide completely validated protocols for every instrument. It's a very standardized solution--a very low cost solution--so people can stop worrying about the details of all of their sample preparation and really focus on the analysis," states GeneMachines' founder, Scott Hunicke-Smith. With the instruments currently avail- able and with several more in the pipeline, GeneMachines is doing just that. GeneSuite comprises seven main components that cover the entire gamut of genomic research: HydroShear™, a novel and highly effective DNA shearing tool; Gel-2-Well™, a fast, high-precision colony and plaque picker; HiGro™, a compact and efficient multichamber incubator; RevPrep™, a flexible and modular DNA purification station; PolyPlex™, a fast, 96-well oligonucleotide synthesizer; OmniGrid™, a sample spotter and microarray maker and TriCycle™, a high-throughput, low-volume thermal cycler. There are also some additional tools, such as HybChambers™ for array hybridization, Pressure Filtration Units, and Array Centrifuges. Shear Madness HydroShear HydroShear is a flow-based DNA-shearing tool used during library construction.2 Its unique design uses the sharp constriction of a ruby orifice to align DNA with the flow path and gently stretch it until it snaps. During repeated passages through the orifice, DNA larger than a certain size (determined by the flow rate and the orifice size) will be snapped, while smaller DNA fragments pass through unchanged. Handling samples as small as 60 µl, HydroShear can load, run, and wash in about 15 minutes. As all HydroShear protocols are computer controlled, day-to-day and user-to-user variations are eliminated, allowing a similar pool of fragments to be created from different starting materials. The sheared DNA has a tight size distribution, with 90 percent of the fragments falling within a twofold range. For example, a target size of 2 kb would produce a pool of DNA fragments with a mean size of 2 kb and 90 percent of all fragments falling between 1.3 and 2.6 kb. Direct cloning of DNA prepared using HydroShear is relatively efficient, although an end repair step increases cloning efficiency. Pickin' and Grinnin' So yesterday you subcloned 1 kb fragments sheared from 20 40-kb cosmid inserts into an M13 vector. Over the next few days you get to pick 1,000 plaques from each library. Lots of coffee and some great tunes on the boom box may help this process, assuming your eyes don't cross too much. Or you can use Gel-2-Well--at about 2,000 picks per hour, you'll be done by suppertime. Gel-2-Well Gel-2-Well is closely based on designs from the Stanford University DNA Sequencing and Technology Center and is centered around a rotating turret of tungsten needles. The plate-handling system can accommodate 72 rectangular petri dishes at once. The plate being processed is imaged by a CCD camera, and the images are analyzed using a PC. For bacterial colonies, the plate is illuminated from above. Colonies show up as bright points against a dim background. A red filter further discriminates blue and white colonies. Switching to plaque counting simply involves changing the light source; plaques are illuminated from behind against a dark background and again, a red filter improves discrimination. A graphical interface allows background subtraction control and selection of features based on brightness, roundness, and separation from other features. Gel-2-Well can pick colonies or plaques as small as 0.5 mm in diameter that are separated by only 0.5 mm. Optimal use of Gel-2-Well requires reasonably dense coverage of transformation plates. If the average number of positive clones per plate is low, plate-handling and imaging time is increased. At 50 positive targets per plate, Gel-2-Well picks around 750 clones per hour, while at 400 targets per plate this improves to more than 1,750 picks per hour. Within this range, if the clones are evenly distributed across the plate, about 90 percent of positive (white) colonies can be recovered, and more than 95 percent of negatives (blue) are rejected. Less than 3 percent of the output cultures will be mixed or failed inoculations. Colonies or plaques are picked from the imaged plate by tungsten needles. Each needle is washed by a high-pressure jet of water, sterilized in a small furnace, and dipped into the next selected feature. The needle is then plunged several times into a microtiter well containing the appropriate growth medium. Gel-2-Well's high throughput is achieved in part by performing all these steps in parallel with different needles on the turret. Gel-2-Well handles 72 microtiter plates at once; this format allows nearly 7,000 unattended picks into 96-well plates. Gel-2-Well can also create arrayed libraries in 384-well format (over 27,000 unattended picks). Software also keeps track of clone placement. HiGro Shake Well The microtiter cassettes from Gel-2-Well are then loaded into HiGro, GeneSuite's high-capacity oxygenated environmental shaker. HiGro accepts four cassettes, each of which contains 12 microtiter plates. The cassettes are loaded into four independent growth chambers. During growth, HiGro controls temperature and oxygen level for each chamber. The chambers can be vigorously shaken at up to 650 rpm on an 8 mm orbit. Combining fast orbital shaking with precise temperature control and increased oxygenation results in reproducibly excellent yields for bacterial and phage cultures. During 20-hour incubations, HiGro can achieve sixfold improvements over microtiter cultures using other systems. Revolutionary Thinking RevPrep Targeted for release in the first quarter of 2000, RevPrep, the Revolution PrepMachine, is a completely automated DNA purification system. It purifies plasmid DNA from cultures grown in 96-well plates for less than five cents per sample. Often DNA preparation robots rely on expensive vacuum filtration dishes or require time-consuming manual centrifugation steps. RevPrep's Array Centrifuge eliminates both of these problems. The Array Centrifuge, also developed at the Stanford DNA Sequencing and Technology Center, features a 96-well format array of individual flow-through microrotors.3 Each rotor is precision-machined in two halves from titanium rod and press-fitted together. The cavity inside each rotor superficially resembles two cones, stacked base to base, with a sample inlet at the point of one and a sample outlet at the point of the other. The rotors are assembled into an 8 x 12 array. The current design achieves equal rotational velocity in each rotor using a direct drive system. The low-mass rotors quickly and safely reach speeds of 70,000 rpm. The liquid in the rotor is self-balancing as it is pushed out into the rotor by centrifugal force, leaving an axial tube of air about the same diameter as the inlet aperture (0.08 in.). This leads to just under 6,000 x g at the liquid surface and 20,000 x g at the widest surface of the rotor--adequate to pellet Escherichia coli from a 400 µl culture in just under one second! Insertion of a cylindrical section inside each rotor, between the bases of the two cones, improves pellet retention during deceleration. With the base-to-base design, bacteria pellet into a thin skin of cells rather than a big clump at the rotor's waist, reducing the shear forces on the pelleted material during deceleration. Deceleration rates of ~500 rpm/second ensure the pellet remains intact. Shear forces can, however, be used to the design's advantage. Rapid acceleration from 0 to 30,000 rpm (about one second for a complete cycle) efficiently resuspends pelleted material. The rotor is finally cleaned using a high-pressure water wash. The basic RevPrep is set up to perform plasmid purifications using an optimized alkaline-lysis procedure. From 400 µl E. coli cultures grown in the HiGro unit in a deep-well microtiter plate, ~10 µg of DNA can be recovered at a final concentration of ~160 ng/µl. The dual Array Centrifuge option performs alkaline lysis purifications in 30 minutes, and DNA purified in this manner can be used for fluorescent DNA sequencing without further purification or concentration. RevPrep will process 16 input sample plates, allowing eight hours of unattended operation. PC control of the Array Centrifuge combined with a 20-500 µl multichannel pipettor allows for several protocols to be automated. A Pressure Filtration Station, faster and more efficient than many vacuum-driven filtration processes, increases versatility. Covering the Bases The PolyPlex is a 96-well synthesizer capable of generating oligonucleotides and other synthetic polymers.4 GeneMachines finalized licensing arrangements for oligonucleotide synthesis with PE Biosystems in November 1999. Each of the 12 reagent systems has its own dedicated delivery manifold; the Stanford DNA Sequencing and Technology Center observes a 10-fold reduction in oligo synthesis costs using this approach. PolyPlex uses phosphoramidite synthesis chemistry, using 96-well filter plates. Computer (PC or Mac) control allows 96 oligos--each of a different sequence--to be synthesized simultaneously. The design controls the synthesis environment and gives acceptable coupling efficiencies of >98 percent. Synthesis of 96 oligos takes less than 10 minutes per cycle, theoretically allowing 20-mers to be synthesized in less than four hours. Oligonucleotide syntheses are on the 20-50 nmol scale, at a cost of less than 10 cents per base. PolyPlex currently allows one unattended run, and four 96-well plates of 20-mers can be synthesized in a day. OmniGrid Array of Options OmniGrid is GeneMachines' high-precision multiaxis microarrayer. OmniGrid can deposit the samples from 96- or 384-well plates onto standard microscope slides or nylon membranes. Four sample plates and 100 microscope slides are currently supported. One 384-well plate can be printed onto 100 slides in 20 minutes using 16 printing pins. OmniGrid can be configured with up to 32 pins. OmniGrid uses high-speed, brushless servo motors to obtain fast, high-precision printing. Printing precision is +/-5 µm. Pins are available to create features as small as 100 µm. MicroQuill split pins (manufactured by Majer Precision Engineering Inc.) are similar in concept to the tip of a fountain pen. A split in the pin shaft holds a small reservoir of printing solution that allows multiple spots to be printed without returning to the reservoir. GeneMachines is also developing a solid pin, called the Spotlight™, which should be more durable and reliably cleaned. Pin cleaning is critical to eliminate spot-to-spot contamination. OmniGrid allows a number of customizable pin cleaning options, based on sonication, water flush, blotting, and vacuum drying, to accommodate a range of sample types. The entire machine is housed in a clear Plexiglas enclosure, allowing complete visibility and stringent environmental control. The air is HEPA filtered to ensure dust-free printing and hygrostatically controlled to ensure constant humidity, improving reproducibility. Microarraying is rapidly becoming a major technique in many research areas, and GeneMachines is planning a number of options to increase OmniGrid's flexibility, including surface-coated slides, bar-code reading, CCD monitoring of print runs for quality control monitoring, and integration with the Automated Server Arm to increase capacity. Slides printed using OmniGrid can be hybridized in HybChambers, simple, low-volume containers for standard microscope slides. Each HybChamber, which is designed to be incubated in a standard water bath or oven, holds two slides, and chambers can be stacked to conserve space during processing. The chambers include a leakproof seal and can withstand temperatures up to 75°C. In the Pipeline GeneSuite's current commercial repertoire lacks a thermal cycler. For many labs, taking output DNA from RevPrep and performing PCR or cycle sequencing in a conventional thermal cycler is not too painful, but to fill this gap, GeneMachines is developing the TriCycle. This machine uses a novel, proprietary method for thermal cycling with the goal of processing more than 10,000 samples per day, with sample volumes of less than 1 µl. In addition to the featured instruments, individual components are sold separately. The Microwell Plate Cassette is a useful accessory for anyone who handles large numbers of microtiter plates, the Pressure Filtration Station is an efficient alternative to the often messy vacuum-driven 96-well plate filters, and the Automated Server Arm can be integrated with most robotic workstations. To recognize the contribution of the Stanford DNA Sequencing and Technology Center in dev-elopment of several of the components of GeneSuite, some instruments also carry Stanford's SUTECH label. "Stanford wants SUTECH to be synonymous with the very best--the hottest--technology to come out of its research programs," said Hunicke-Smith. While GeneSuite is a defined collection of instruments, many are modular in concept and can be provided with a number of different options. Further, GeneMachines' collected biological and engineering expertise allows it to offer a variety of custom system analysis and engineering solutions. Bob Sinclair can be contacted at bobsinclair@tech-write-edit.com. References 1.M.D. Brush, "Liquid handlers have the starring roles in high-throughput robotics," The Scientist, 13[4]:22, Feb. 15, 1999. 2.Y.R. Thorstenson et al., "An automated hydrodynamic process for controlled, unbiased DNA shearing," Genome Research, 8:848-55, 1998. 3. A. Marziali et al., "An arrayable flow-through microcentrifuge for high-throughput instrumentation," Proceedings of the National Academy of Sciences, 96:61-6, January 1999. 4. D.A. Lashkari et al., "An automated multiplex oligonucleotide synthesizer: development of high-throughput, low-cost DNA synthesis," Proceedings of the National Academy of Sciences, 92:7912-5, 1995. For More InformationGeneMachines(650) 508-1634www.genemachines.com

Suite Dreams

Interested in reading more?

Become a Member of