Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Even though PCR has been around for decades, finding a PCR instrument is not necessarily a simple task. Today there are many decision points to navigate (what kind of PCR? what kind of format and throughput?), so an overview of the choices may be helpful as a roadmap. Here are some important features of PCR instruments to consider when navigating through the options and some examples of new developments in PCR technology.
Methods of PCR
There are three main methods of PCR: standard (or endpoint) PCR, quantitative PCR (qPCR) and digital PCR (dPCR). Although they all amplify DNA, they differ in their methods, applications, throughput levels and sensitivity and in some instances use different instrumentation.
Standard PCR
Standard PCR is used to amplify a particular sequence, such as when more of a sequence is needed as a reagent for an experiment. This is also called endpoint PCR, because it is focused on obtaining the end product.
Quantitative PCR
Quantitative PCR typically is used to measure the relative abundance of a particular target sequence. It is also called real-time PCR, because the extent of the reaction is monitored as it occurs using fluorescently tagged bases (unlike endpoint PCR). Also in contrast to standard PCR, the absolute amount of a sequence can be calculated from qPCR by running a standard curve for comparison. Advantages of qPCR include the ability to quantify a target sequence (though not as sensitively as dPCR) and opportunities for higher throughput compared with dPCR.
Digital PCR
For digital PCR, the sample reaction is partitioned into a large number (hundreds to millions) of very small volumes before amplification. Because the partitions are so minute, each one typically contains either zero or one copy of the target sequence. After amplification, the number of partitions containing target sequences is counted to obtain an absolute measure of the original sequence. An advantage of digital PCR is that it is more sensitive than qPCR—as such, it’s well suited for quantification of rare sequences, copy number variation analysis and gene-expression analysis of rare targets. This method can also distinguish between copy numbers with better resolution than qPCR. A potential disadvantage of dPCR is limited throughput, compared with qPCR.
Sample formats
The standard format for PCR reactions—96 wells using 0.2-ml tubes—has been around for a while, but today many more options are available. Tube sizes generally range from 0.1 to 0.5 ml. Reaction volumes range from 5 to 100 µl. The number of tubes per run also varies, with blocks ranging from 48 to 1,536 wells. Tube strips and plates are also available. The organization of sample blocks can be determined by the user—for example, some PCR instruments offer interchangeable blocks that can be swapped to change formats; they can even be combined and independently controlled for different PCR reactions running concurrently. QIAGEN’s Rotor-Gene Q qPCR instrument uses interchangeable rotors that can hold 36, 72 or 100 samples. “Rotors spin in a chamber of moving air, keeping all samples at precisely the same temperature during thermal cycling,” says Jodi McBride, global product manager in instrument marketing at QIAGEN.
Throughput
The standard 96-well, 0.2-ml tube PCR format is considered low to medium throughput today, which for many experiments is more than sufficient. But additional options are appearing for those who want to increase their throughput, running up to 1,536 samples simultaneously. In addition, still greater throughput can be achieved by linking individual instruments together. For medium to high throughput, the “Mastercycler pro can connect one to five units with a control panel, or up to 30 units with CycleManager pro software,” says Jessica Geisler, product manager for thermal cyclers at Eppendorf. The Mastercycler nexus can connect one to three units for low to medium throughput.
Barcoding sample plates, in combination with automated, robotic handling systems to move plates around efficiently, is another way to boost throughput. Bio-Rad Laboratories plans to release a new version of its CFX Automation software that “will offer more flexibility in the use of barcodes, such as accommodating special characters,” says Pete Skirpstunas, product manager in gene expression at Bio-Rad. Multiplexing PCR reactions—though tricky and requiring careful optimization—is another option for running more experiments in less time.
Ease-of-use features
Instruments differ in some of their features—which may or may not appeal to users, depending on their type of research—but most features aim to enhance ease of use. An exhaustive listing isn’t feasible, but examples include “a user-specific QuickStart feature and a multiblock system with quick block exchange,” says Alexander Berka, general manager of the life science business unit at Analytik Jena. Eppendorf offers patented lid features: The VapoProtect lid on the Mastercycler pro minimizes evaporation from samples, and the flexlid on the Mastercycler nexus automatically adjusts its height to fit a variety of sample tubes and consumables.
PCRmax also incorporates ease-of-use features such as USB logins, which might be especially appreciated in large, busy labs. Each user’s USB key acts as a login stick, according to Andrew Birnie, business development specialist at PCRmax. “When they insert it, the Alpha Cycler will display only their programs and not allow anyone else to access or modify them,” he says.
Another ease-of-use feature to consider is the ability to control the PCR machine remotely. For example, Agilent’s SureCycler 8800 PCR instrument offers “remote access to start, stop and monitor experiments from most web-enabled devices and is a feature that will be available soon on the AriaMx qPCR instrument, as well,” says Anantha Poluri, product manager for PCR and qPCR at Agilent Technologies.
Software
Another key factor to consider is the software that runs the instrument during the experiment, and which is often used afterward for data analysis. You want to find software that can do the analysis you require, but ease of use is also crucial—especially in labs with a mix of beginners and experts. For example, Bio-Rad Laboratories released the CFX Automation II system with an emphasis on accessibility. “The CFX Automation II system makes high-throughput PCR a reality for users who don’t necessarily have knowledge of scripting,” says Skirpstunas.
QIAGEN will release new operating and analysis software this month. Called Q-Rex, the software is designed to help researchers at all experience levels with setting up assays and analyzing data. “[It] includes wizard-based designs, making it suitable for use by the novice researcher, while maintaining the highly complex data-analysis features required by advanced researchers,” says McBride.
PCRmax’s Alpha Cycler is run by software that transmits a quick-response (QR) code to users when scanned by a mobile device using a free app. This gives the user valuable information, such as the time needed to finish a run. But the app also sends the information to PCRmax, because “in that QR code are health-check codes that monitor the performance of our systems,” says Birnie. “They allow us to spot potential problems to ensure that the systems won't let researchers down.”
Above all, a PCR machine’s ability to heat evenly and accurately is key for sensitivity and reproducibility. “Variations in thermal uniformity can lead to mispriming and misamplification events along with detrimental effects to enzyme performance, which can alter data quality and reliability,” says McBride. Thankfully, many PCR instruments that are available today meet this criterion. All that’s left is finding a system with features—and accompanying software—that best suit you and your research.