Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Thermocyclers (aka thermal cyclers or PCR machines) have come a long way since the first prototypes nearly three decades ago, taking their place in the lab as the go-to tool for everything from genotyping to DNA sequencing library preparation to cloning vectors and plasmids. Today’s instruments are faster, and most achieve about the same level of thermal performance. “All can do basic PCR pretty well,” says Mike Mortillaro, owner of Bulldog Bio, a Portsmouth, NH-based provider of products for the life sciences. “Thermocyclers are now seen as commodity products.”
There’s a general trend toward making the instruments easier to use, too, with far more options available today than just a few years ago—not just in terms of vendors, but in the way samples can be introduced, the number of possible samples, how the instrument is controlled and even whether to run gradients or multiple different programs simultaneously.
That said, purchase of traditional thermocyclers has dropped off rapidly in the past three years for a couple reasons, Mortillaro says. First, a glut of thermocyclers has been created as next-gen DNA-sequencing technologies have lessened the demand for cloning. Second, the advent of qPCR techniques has lessened the popularity of endpoint PCR as an informational tool.
Still, if your lab is in the market for a PCR instrument, here are a few variables to consider.
To Q, or not to Q
qPCR is similar to endpoint PCR in that both use a DNA polymerase to exponentially amplify DNA during thermal cycles by elongating a primer bound to a template. The main difference is that endpoint PCR generally identifies the PCR products by gel electrophoresis after the cycles have completed, and qPCR detects amplicons while the reactions are proceeding as an accumulation of fluorescent signals (thus its more complete name, “real-time quantitative PCR”).
Because qPCR is quantitative, it can look not only at whether a particular DNA sequence is present in a sample, but also at whether it is up- or down-regulated, or how many copies of it there are to start with. (In contrast, traditional endpoint PCR is not quantitative. The intensity of a band on a gel is not necessarily indicative of the relative abundance of that target in the starting sample.) So although the upfront cost of qPCR is considerably higher, it also can answer questions that a standard thermocycler cannot.
There are a host of considerations having to do with qPCR’s fluorescence detection capabilities—such as filter sets, number of channels for multiplexing, sensitivity and dynamic range as well as programming—that are not relevant to a broader discussion of thermocyclers. Generally speaking, however, qPCR instruments are distinct from endpoint thermal cyclers. But at least one manufacturer, Bio-Rad Laboratories offers an upgrade path from PCR to real-time PCR by supplying a separate optical module for the C1000 Touch thermocycler chassis.
Formats and flexibility
Different instruments—or at least different configurations of heating blocks on the instruments—handle different numbers and types of samples. The first thing to decide is how many wells you need, and whether you plan to use PCR tubes, plates or strip tubes (which are like tubes attached together in the format of a plate), says Graziella Mendonsa, product manager at laboratory product and supply distributor MIDSCI. It’s also important to know whether you’ll be using low-, regular- or high-profile tubes, skirted or un-skirted plates or even microscope slides.
Some configurations can accommodate a variety of formats, and others are much more restricted. Although some instruments let you switch out blocks (which hold and heat the sample), most don’t, says Peter Skirpstunas, senior product manager for thermal cyclers at Bio-Rad. He queries, “How confident are you that six months from now you’re not going to need a different block format? How much flexibility will you need in the instrument?” Bio-Rad’s C1000 Touch and S1000 instruments can be outfitted with any of four standard PCR blocks—a 96 fast, 96-deep well, 384-well and dual 48-well blocks (in addition to three real-time modules available for the C1000 Touch). The cost of an additional block, says Skirpstunas, is less than that of an entire new system.
The advent of divided blocks, notes Emir Hodzic, director of the qPCR core facility at the University of California, Davis, School of Veterinary Medicine, lets users “set up one part of the block at one annealing temperature and another one to another annealing temperate.” In fact, the individual blocks can generally be independently started, stopped and controlled. Many vendors offer such instruments—for example, the Applied Biosystems Veriti® thermal cycler from Life Technologies can be equipped with six blocks—turning a single PCR machine into what amounts to multiple instruments occupying a single space.
Gradients
Even if you choose not to purchase a multiple-block instrument, you may still be able to simultaneously run different reactions at different temperatures. Multiple manufacturers offer instruments that allow the block to be heated not uniformly but as a precisely defined gradient, which enables you to very quickly pick the best temperature. “You can run one experiment using eight different annealing temperatures, and then run them on a gel to see which one works the best,” points out Skirpstunas. “It’s a quick, easy, cost-efficient tool for assay optimization.”
It may even be possible to add gradient capabilities after the fact. Bibby Scientific’s 3PrimeX and Prime units are sold as standard, nongradient instruments, for example, but can be upgraded with software supplied on a USB stick, says Mendonsa.
Communications
Many vendors now offer touch-screen; ATM-style, menu-driven; or other easy-to-use control screens, says Mortillaro. Indeed, there has been a general trend over the past few years of making the instruments easier to use, says Skirpstunas, including at Bio-Rad, which has transitioned its thermal cyclers to touch-screen interfaces.
Many manufacturers offer networking capabilities, as well. Some instruments can be controlled remotely through an Ethernet connection. Some can be networked together—which is especially useful for higher-throughput applications—and all controlled from a single instrument or from a PC. And at least two systems—the Agilent Technologies SureCycler 8800 and Life Technologies’ ProFlex—offer the ability to monitor the instrument wirelessly using a smart-phone app.
Final considerations
Some users are looking to do “fast PCR,” and some manufacturers tout their instrument’s ability to facilitate this. But the speed at which PCR can be performed is as much a function of the enzyme and plasticware as it is the instrumentation, points out Ossian Saris, an application specialist at Thermo Fisher Scientific. In addition to rapid ramp rates, Thermo’s Piko thermocyclers use dedicated tubes and plates with ultrathin walls that transfer heat more rapidly between the block and the sample.
Saris notes that a unit’s footprint and the noise the instrument makes also can be issues for already crowded labs.
A relationship with a vendor, too, can be an important criterion in deciding on a purchase. Erin O’Leary-Jepsen, managing director of the molecular research core at Idaho State University, says she will be purchasing a new instrument this month from Bio-Rad, adding to the two MJ Research (now Bio-Rad) instruments her facility currently has. She relates the reason for selecting Bio-Rad to Biocompare in an email: “Vendor – reputation, customer service, instrument quality and reliability, sales rep’s willingness to listen to our needs and negotiate the best price for us, warranty/service contract – this is huge for us, as a longer warranty period postpones the need to purchase annual service contracts.”