High-Throughput Thermocyclers

Photo: Courtesy of Applied Biosystems The Auto-Lid Dual 384-Well GeneAmp PCR System 9700 from Applied Biosystems. Used to be, researchers used thermocyclers for PCR. But times have changed. Once called PCR machines thermocyclers are now required for a wide range of common applications such as sequencing and genotyping. Murray Anderson, director of core PCR for Applied Biosystems, Foster City Calif., observes, "When we first launched the dual 384-well [thermal cycling] systems, the primary customers were users doing sequencing. ... We have [recently] seen an expansion in numbers of people doing genotyping." And that's not all: The rate of throughput is changing, too. Academic labs can now process samples at rates previously unheard of outside of large sequencing labs. Yet, for all the bells and whistles, these instruments remain thermocyclers, and when it comes to making a purchasing decision, accuracy, speed, format, and reliability are still the most important considerations. Sample format is perhaps the most important criterion. Options include tubes, microplates, and thin-walled capillaries. Thermocyclers that heat by contact between the sample and a solid metal block can often accommodate either tubes or microplates; multiple interchangable blocks can accommodate a variety of sample types in these instruments. The once-standard 0.5-ml tube format is now inconvenient for all but the lowest throughput tasks and is increasingly uncommon, having been supplanted by blocks that support 0.2-ml tubes. The form factor of these tubes is identical to that of the wells in a 96-well plate, and the common 8 x 12 grid of a 96-well system can support both tube formats. Instruments that can accommodate 384-well plates are generally dedicated high-throughput devices, as they are designed specifically for use with these microplates, which is an inconvenient format for a small number of reactions. Capillary tubes have far better thermal properties and a greater sample surface area than plastic microwell plates; these qualities facilitate rapid heating and cooling cycles. The RapidCycler from Salt Lake City-based Idaho Technology is a capillary-based system that can handle only a small number of samples per run, but it still qualifies as a high-throughput instrument because of its rapid cycle times. The RapidCycler can perform 30 amplification cycles on a short PCR product in 15 minutes with a maximum of 48 samples per run. "The real trick is in the capillaries," says marketing associate Mark Kessler. "In a capillary, the liquid is spread over the area of the tube, giving it a larger surface-to-volume ratio." But capillary-based systems have their limitations, too. The capillary tube must be heat-sealed before running the reaction and broken open afterwards to access the sample. Also, preparation using multlchannel pipetters is inconvenient. CONSIDER AUTOMATION Once sample format has been determined, researchers with very high-throughput needs may want to consider automated systems to maximize productivity. Al Outhouse, senior applications scientist for Burlington, Canada-based Thermo CRS, observes that "the traditional calculation for automation is the cost of the robot versus the cost of having a technician perform the task. Also, robots can do things technicians can't do, like run 24 hours without breaks." The decision to purchase a robot can have an impact beyond raw throughput. As David Sands, CEO of Trenton, NJ-based ST Robotics International points out, "One thing the robot system gives you is consistency. If people are moving things from one place to another they do it differently every time. With a robot system you start it all going, you leave the room, and it does it the same every time." The easiest way to integrate a robot into a thermocycler system is with a motorized lid. Automated operation of the lid facilitates robotic removal of one plate and replacement with the next in line. The robotic systems used are quite versatile, and a complete system may include equipment for plate sealing, automated liquid handing, and refrigerated storage in addition to thermocycling. After all, "The actual thermocycling itself is not a speedy process, so generally speaking you don't get a lot of benefit from automation," says Jim Schools, director of marketing at Springfield, NJ-based Hudson Control Group. "Where automation comes into play more often is that there are pre- and post-PCR steps that involve other pieces of equipment. ... Tying together the multiple devices is where robotics really gets involved." Photo: Courtesy of Thermo CRS ARMED AND READY: Thermo CRS Catalyst 5 robotic arm loading a bank of MWG high-throughput thermal cyclers. MULTIPLE OPTIONS A complementary approach is to purchase a system with higher capacity. Researchers can easily increase throughput from 96 wells per run to 384 by changing sample format. When constructing a system larger than the 384-well block level, however, users have several options. The soon-to-be-released MatriCycler (below) is expected to support 1,536-well plates. Alternatively, researchers may purchase and network multiple independent systems; buy one or more multiple block-integrated systems; or construct multiple-block modular systems. The simplest approach is to link multiple single-block systems with networking software. Techne of Princeton, NJ, adopted this approach with its FlexiQuad system--essentially four FlexiGene systems linked through a single PC. Todd Kwitchoff, Techne's life sciences product manager, notes an advantage: if "one [thermocycler] goes down you can still use the other three" rather than potentially losing the entire system. Likewise, Waltham, Mass.-based MJ Research's thermocyclers can be networked using the Easy Engine software option in any combination of Tetrad (four-block), Dyad (two-block) and one-block DNA Engines. Cambridge, Mass.-based Intelligent Bio-Instruments' TC1600 is a representative multi-block integrated system. The instrument is sold as a set of 16 blocks, controlled in groups of four. The dual-block version of the Applied Biosystems GeneAmp PCR System 9700 is also a multi-block system, as are MJ Research's Tetrad and Dyad systems. MJ Research is releasing another system add-on, the Dyad Disciple, which will add two blocks to an existing Dyad, effectively turning it into a Tetrad. Each MJ system will work with the company's interchangeable sample blocks, called Alpha™ units, increasing the systems' modularity. The Primus Multiblock by Ebersberg, Germany-based MWG Biotech and the Multi Block System (MBS) by Franklin, Mass.-based Thermo Hybaid take yet another approach by stressing modularity. These systems are designed to accept blocks in sets of one for the MBS and in sets of one, two, or four for the Primus Multiblock. Both systems provide one power supply per block (Thermo Hybaid's power supply is fully integrated into the unit), are shipped with a preconfigured PC, and are easy to automate. The motorized version of the Primus Multiblock, for instance, has extra space milled out of the block to simplify robotic gripping. Osama El-Badry, field applications manager for Thermo Hybaid, notes that the MBS features a CD-drawer mechanism to give the robot easy access to the block. He adds that MBS units are also stackable, which reduces space requirements. At the other end of the scale, an increasing number of labs have one-time or occasional high-throughput needs, for which purchasing a new instrument or even a new block for an existing instrument may be prohibitively expensive. For these labs, adapting existing resources may make more sense. For instance, specialty plates such as the Split Well CyclePlate®-192ET and -384ET by Hudson, NH-based Apogent Discoveries provide additional capacity per run. These plates follow the standard 96-well configuration for compatibility with standard thermocyclers, but each well is split into halves or quarters to enhance the number of simultaneous reactions. HEATING AND COOLING All thermocyclers require an efficient means of heating and cooling their samples, and all accomplish it in the same way: by contact between the sample and a material at or near the desired temperature. Solid media, such as aluminum or silver blocks, are commonly used for Peltier-based cyclers, while other systems utilize circulating water or forced air to modulate sample temperature. Air- and water-based thermocyclers are less common in the current market. The BioOven III by St. John Associates of Beltsville, Md., uses rapidly circulating heated air and a rotating sample holder to heat and cool samples, and is capable of processing up to 10 standard plates at once, according to President Peter St. John. This approach has the advantage of sample format flexibility, but its main attraction is high capacity with a very low price tag. The DT-24 and DT-108 water-based thermocyclers marketed by Marsh Bio Products of Rochester, NY, also provide high throughput at a lower price. These instruments can process up to 24 or 108 plates simultaneously, depending on the model. Dave Witkoski, Marsh's director of marketing, says that while the "water bath isn't a new technology ... [it is] a good mechanism for doing a lot of plates at once" and provides an "even distribution of heating over the whole plate." The majority of thermocyclers on the market today, high throughput or otherwise, are driven by Peltier-effect heat pumps. Named for French physicist Jean Peltier, these systems operate differently than do the vapor compression heat pumps found in air conditioners and refrigerators. Essentially, applying a current through two conductors with dissimilar electron densities causes heat to be absorbed on one side and released on the other--the reverse of the better-known thermocouple effect. By controlling the direction of electron flow, such a device can act as both a heating and cooling mechanism. Peltier originally used different metals, but modern instruments use semiconductors arranged in small pairs of blocks and sandwiched between layers of ceramic. Each block is a separate Peltier element, and the presence of many small blocks helps to ensure an even temperature distribution. Some companies have tweaked Peltier's original design. According to Hercules, Calif.-based Bio-Rad's amplification product manager Steve Gagliardi, the Peltier-Joule technology used in a Bio-Rad iCycler "still uses Peltier modules but also uses resistive heating elements to ensure [temperature] uniformity, especially on the outside set of wells." Spokane, Wash.-based MatriCal offers another variation. The company's Matri-Cycler, currently in beta testing and scheduled for release in the first quarter of 2003, inverts the typical process, says President Kevin Oldenberg. "You have a standard plate and a lid that sits on the plate. Pins insert through the lid and into the liquid sample. We bring the Peltier in contact with the pins, which heats and cools the pins ... [and] samples." The advantage of this system, says Oldenberg, is that "standard thermocyclers use an aluminum block for heating and cooling. From a purely physical standpoint, that's very ineffective, because you are trying to [heat and cool] a thermal mass [many times] larger than the thermal mass of the sample. ... Also you are driving heat across the plastic, and plastics are among the best known insulators." The Robocycler by La Jolla, Calif.'s Stratagene takes an entirely different approach to thermal cycling. Instead of changing the temperature in a single block, the Robocycler moves the samples between four constant temperature blocks using a simple robotic arm. Such an approach obviates such concerns as ramping speed, but also prevents researchers from using programs that change the annealing temperature for each cycle. Photo: Courtesy of ST Robotics International ST Robotics International's small genomics system (top) and large-scale high-throughput screening system (bottom). MAKING A DECISION When comparing thermocyclers, as with any other large purchase, the devil is in the details. The published specifications should be carefully considered, but a hands-on test is the best method. "Consumers need to note the specifications and ask themselves 'How are they measured by the manufacturer?'" says Thermo Hybaid's El-Badry. "It's important for the manufacturer to stand by their claims, but it's also important for the customer to run tests in their own laboratory to be sure the cycler performs to the required specifications"--a sentiment echoed by many of the company representatives interviewed. For example: Ramping speed is not linear, and the specifications do not typically report whether the ramping speed is an average or a maximum. Nor do they specify the temperature endpoints between which the measurement was taken. Similarly, carefully designed heated lids are extremely important, especially with 384-well models, and need to be tested. As MWG Biotech's Abrahams points out, "In a 96-well format you don't notice evaporation [very much] ... but with 2-3 microliter reactions [as in the 384-well system] evaporation becomes an issue." The purchasing decision may hinge on a unit's expandability or special features, such as the ability to perform real-time PCR. Indianapolis-based Roche Applied Science's LightCycler, a capillary-based system, offers built-in real-time PCR capability, whereas BioRad's iCycler offers this function as an optional add-on module. Other bells and whistles include MJ Research's Remote Alpha Dock, which allows an Alpha block to be operated some distance from the Tetrad base. Another is the backwards compatibility mode of the Applied Biosystems GeneAmp 9700, which replicates the thermal profile of the company's 9600 model. Finally, consider the system's user interface, as these can vary considerably. Most small systems rely on small monochrome interfaces, though the iCycler and Dyad, for instance, have 1/4 VGA (320 x 240) screens. Other systems come complete with a computer system and accompanying software, easing both system control and reaction monitoring. Jeremy Peirce (jlpeirce@princeton.edu) is a freelance writer in Jersey City, NJ.

High-Throughput Thermocyclers

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Photo: Courtesy of Applied Biosystems
The Auto-Lid Dual 384-Well GeneAmp PCR System 9700 from Applied Biosystems.