Amplify, Detect and Quantitate with These PCR Options

PCR in its various forms has been a mainstay in the molecular biologist’s toolbox nearly since its discovery by Kary Mullis more than three decades ago. The fundamental principle—exponential amplification of a sequence of DNA by reiterative use of a template-based polymerase—has remained steady through vast improvements in reagents, protocols and instrumentation, which have in turn given rise to corresponding leaps in speed, fidelity and the ability to multiplex, quantitate and more.

Depending on their needs, today’s researchers may run a few samples in a basic (endpoint) thermal cycler and visualize the results by gel electrophoresis—perhaps to verify the existence of a sequence in the original sample or maybe to cut out the band and use it for cloning or another downstream application. Or they may run a few hundred samples in a real-time quantitative PCR (qPCR) instrument for genotyping, with status of multiple alleles graphically displayed. Or perhaps they quantitate the rare needle in a haystack using digital PCR (dPCR).

Here we look at some key features of today’s (and tomorrow’s) PCR instrumentation that facilitate ever-easier and more streamlined data generation, collection and analysis.

In common

There are many aspects of endpoint PCR, qPCR and dPCR instrumentation that can be considered and discussed in common. 

Broadly speaking, ease of use is among customers’ paramount concerns.

This may include, for example, instrument setup and experimental setup, the number and availability of pre-programmed protocols or a wizard for customizing and editing protocols, notes Caroline Tsou, director of global product marketing for life sciences at Agilent Technologies.

The progression of thermocyclers provides researchers with many options. Older and legacy models may offer different ways for customers to interface with the instrument vs. cutting-edge models; personal or budget systems may differ from those built for a multiuser or a regulated environment. Newer and higher-end instruments often offer touch-screen control and monitoring, in some instances with remote connectivity. The latter may include not only control and run-progress reporting but also functions such as error tracking and diagnostics.

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Customers look for “meaningful feature sets—things that are actually useful,” says Andrew Birnie, life science product manager at Bibby Scientific. “In this category would be things like an ability to lock protocols so that no one else can adjust them for you, and having a way of tracking the unit” such that the instrument “stores and tracks its own error message[s], or it communicates them out.”

The number of samples that can be run differs greatly. Small, individual, one-sample-at-a-time instruments exist or are being developed for clinical labs and point-of-care situations, notes Linda Cook, director of molecular (PCR) virology in the Department of Laboratory Medicine at the University of Washington and a research scientist at the Fred Hutchinson Cancer Research Center. “But there’s also development of the bigger blocks, as well,” she says.

Throughput

Throughput is about more than just how many samples can be processed simultaneously. Instruments may have a “fast” mode in which, for example, a run takes 40 minutes rather than two hours, allowing a lab to get results back to clinicians that much faster. Speed can be advantageous even in a research core setting, says Anne-Marie Girard, senior faculty research assistant at the Oregon State University Center for Genome Research and Biocomputing: “If you have a lot to do, you can get through a lot more.” But Chandana Batchu, Bio-Rad’s product manager for real-time PCR systems, points out that “we haven’t seen much demand for faster PCR,” adding that “of course, there are special cases.” 

Closely related to throughput are considerations of sample format and size.

Restricting the conversation only to standard formats, samples are typically prepared and run in either tubes or multiwell plates (with options such as microscope slides sometimes available, as well). Instruments can be purchased in a specific configuration—dedicated to 96-well plates, for example. In many instances, the blocks are interchangeable, enabling users to “just change out the block from a 96-well to a 384-well instead of purchasing two instruments,” notes Tsou.

Versions also can be found with up to four blocks. Birnie finds that because control of each block is “utterly independent,” these units are more often than not found in multiuser environments: “Instead of getting six or seven machines, you just get one big multiblock unit, and it gives everyone their own PCR machine contained within that one jacket.”

Multiblock machines also can be run as if they contain a single block, allowing for a corresponding increase in throughput—equivalent to daisy-chaining multiple instruments together, a strategy followed by several vendors.

In addition to accommodating more samples, the 384-well format takes less volume—of reagents as well as samples—so it’s actually cheaper to use. “But if you scale it back too far, then you lose sensitivity, because you’re only able to put so much of the patient sample in there,” notes Cook. “So you have to balance those two things.” And Girard points out that loading the wells by hand “becomes a bit tedious, and you have to be a lot more careful—so the 96-well is easier for an individual” without the aid of robotics.

Just qPCR

Many aspects that might be decision points regarding qPCR (or dPCR) may not be (as) relevant to endpoint PCR.

Some important features to consider when selecting a methodology are temperature control, color channels and data analysis.

Endpoint PCR is most often used to generate a yes/no answer, and so temperature accuracy and uniformity may not be quite as crucial. “But in the qPCR world, it’s quantifying, getting an exact answer,” explains Birnie. Even small variations—both well to well and run to run—“in an enzymatic process like PCR can have a massive impact on the outcome.”

qPCR instruments generally have two to five (or more) color channels. “We have some very sophisticated customers that do make use of the full four- to five-color range, and that’s because they have designed highly multiplexed assays,” says Batchu. Yet most are only making use of two or three channels, so “that’s not really an issue.”

Some instruments now let users save a standard curve, “so you don’t have to keep running standards over and over again,” says Cook. Many instruments also show curves on the instrument in real time, as they’re being generated. But for the most part, any numerical analysis is performed on an associated desktop computer or perhaps in the cloud. “You need to do the whole normalization process, so you need to select a reference gene and a control sample, etc.,” Batchu says, noting that the workflow is “a fairly hefty process.” Although the user interaction may differ greatly among vendors, Cook says that qPCR software’s capabilities—what you can do, what you can look at, what you can print out—haven’t changed much, and most vendors offer similar packages.

Thus user experience—how the instrument is set up and operated; how data is collected, stored and analyzed; and the availability of training both to get up to speed and to efficiently generate meaningful data—joins specifications such as cost, number of blocks, number of samples and thermal accuracy as an important consideration in choosing the appropriate instrument for your research needs.

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