2016 PCR Buyer's Guide

Proven Methodology Expanding Life Science Discoveries and Applications

Introduction

Invented in 1983 by Kary Mullis, the polymerase chain reaction (PCR) enables researchers to expand a single piece of DNA into millions of identical fragments. As a result, the technique makes it possible to detect and study sequences that might otherwise be undetectable, and researchers from across the biological sciences have embraced the concept.

The basic technique couldn’t be simpler. (Mullis famously devised the concept as he negotiated a winding California road en route to his weekend cabin.) All you need is a pair of short, inward-facing DNA oligonucleotide “primers” that flank the sequence to be amplified and a temperature-controlled incubator to run the reaction (a thermocycler).

In the first step, the DNA to be amplified (the template) is heated to near boiling to separate the strands. The reaction is then cooled so the primers may find and bind to their target sites, at which point the temperature ramps up again so that a thermostable DNA polymerase can extend the primers. These three steps—denaturation, annealing and extension—constitute one “cycle,” and there may be 20 to 40 in a complete PCR reaction.

The process may not sound all that different from any other DNA-polymerization reaction, but it is: Because not one but two primers are used, and those primers are facing towards each other, the amount of target DNA in the reaction effectively doubles with each cycle.

Thus, a single piece of DNA can be copied 2^ⁿ times, where n is the number of cycles.

That’s true in theory, at least. The first few reaction cycles don’t actually double DNA, and reagents can become limiting later in the reaction, so actual yield is often lower. Still, PCR sometimes is likened to a genetic copying machine.

In 1993, when Mullis won a Nobel Prize for his discovery, the press release announcing the award noted, “The method offers new possibilities particularly in medical diagnostics, and is used, for example, for discovering HIV virus or faulty genes in hereditary diseases. Researchers can also produce DNA from animals that became extinct millions of years ago by using the PCR method on fossil material.” Since then, the applications of PCR have—like DNA—only amplified, and the technique is now used in fields from forensics and genetics to metagenomics and infectious diseases. Here, Biocompare provides an overview of the key features in the evolution of PCR instrumentation, methodologies and applications.

PCR methods

Researchers have over the years developed an array of variations on the fundamental PCR principles, but the vast majority fall into three basic categories: endpoint, qPCR and dPCR

Endpoint

The original and simplest form of the method—and one still used widely today—is endpoint PCR. Here, the reaction proceeds for some number of cycles until it terminates, at which point the products are examined via gel or capillary electrophoresis or using some comparable method.

Endpoint PCR is not, on its own, quantitative; the amount of DNA the reaction produces does not necessarily reflect how much was present at the outset. Different samples and sequences may amplify with different efficiencies, for one thing. Plus, the nature of the enzymatic reaction is such that, at some point, reagents can become limiting.

qPCR and dPCR

Researchers have devised two basic strategies to overcome challenges when trying to perform quantitative analysis. In quantitative real-time PCR (qPCR), the reaction is altered to include a fluorogenic probe, such as an intercalating dye or a TaqMan probe. But monitoring fluorescence intensity over the course of the reaction, researchers can easily compare the levels of DNA in two or more samples. Digital PCR (dPCR) partitions the reaction into many thousands of subreactions, such that, on average, each contains just one or zero copies of the template. By counting the number of positive and negative reactions at the end of the run and applying a Poisson correction, researchers can determine the number of template molecules in the starting sample.

Though both are capable of quantification down to one or a handful of DNA copies, qPCR and dPCR differ in important respects. qPCR, for instance, provides only relative quantitation—that is, that sample A contains twice as much target sequence as sample B—unless a standard curve is generated; dPCR is absolutely quantitative on its own. On the other hand, qPCR is more mature and well known, with millions of predesigned assays commercially available; dPCR is relatively new and thus less widely available and more expensive. qPCR is also more amenable to high-throughput analysis, and it works over a wider dynamic range 

Significantly, dPCR is generally considered more precise: Whereas qPCR can easily distinguish two-fold differences in concentration—the difference between five and 10 copies, for instance—it struggles with subtler differences. In contrast, dPCR can in theory differentiate five gene copies from six, a difference of less than 20%. That kind of accuracy can be particularly useful when quantifying, say, the number of gene copies involved in a chromosomal rearrangement or the abundance of circulating biomarkers in a cancer patient’s blood. And because the sample is diluted as it is partitioned, and it is read only at the end of the reaction, dPCR also is more robust against inhibitors than qPCR.

Table 1: PCR methods and applications

Instrumentation

Generally speaking, PCR machines come in two formats. The majority of PCR instruments use metal blocks with drilled sample holes, which are heated and cooled via a Peltier heating element. A few, including QIAGEN’s Rotor-Gene Q and Roche Diagnostics’ LightCycler® 2.0, incorporate a spinning rotor in a temperature-controlled chamber, ensuring temperature uniformity via the movement of air and faster ramp rates.

Whatever the format, when purchasing an instrument, there are a few key variables to consider. Perhaps the most critical is sample format and throughput.

Think about format

PCR and qPCR can be performed in Eppendorf tubes, tube strips, slides, microtiter plates (96, 384 or 1536 wells) or microfluidic devices (384-well TaqMan array cards from Thermo Fisher Scientific or integrated fluidic circuits from Fluidigm, for instance), as well as in some more esoteric formats, such as Thermo Fisher Scientific’s 3,072-well OpenArray®.

Though some systems lock users into a particular supported format, others allow users to swap sample blocks (or rotors) to accommodate different sample sizes. QIAGEN’s Rotor-Gene Q system supports different rotor styles for 36 or 72 tubes or a rotor disc for analysis of up to 100 samples. Bio-Rad Laboratories’ C1000 Touch® thermal cycler does likewise for different microtiter-plate formats, including modules for 2 x 48, 1 x 96 and 1 x 384 wells. But while higher-end Thermo Scientific QuantStudio systems enable similar flexibility, the QuantStudio 3 and QuantStudio 5 instruments do not; users of those systems must decide in advance which block format they want at time of purchase.

Assay format flexibility helps users future-proof their investment, to some extent.

A tube-based system may suffice today, for instance, but if you anticipate needing to ramp up, in a few years, to 96 or 384 assays at a time, it’s good to know that’s an option. Similarly, core facilities and other shared resources would likely benefit from the flexibility to support different types and scales of research projects.

There are other mechanisms for boosting throughput, as well. Some systems, for instance, let users join multiple systems together under one control system. Such systems include Bio-Rad’s C1000 Touch, which can string together up to 32 units under the control of one PC. Some systems (such as the QuantStudio 6 Flex and Roche’s LightCycler 1536) also are compatible with laboratory robotics. And some systems also help users get more from their runs by enabling them to program different temperatures on the same block (a concept that Thermo Fisher, which offers such capability on its QuantStudio 3 and QuantStudio 5 systems, as well as on its Veriti® thermal cycler, calls VeriFlex™ temperature control).

Multiplexing

Also critical, for qPCR instruments, is multiplexing capability—that is, how many different fluorescent colors (i.e., amplicons) can be detected simultaneously per well. Bio-Rad’s CFX96 Touch™ and CFX384 Touch real-time systems support six and five fluorescent channels, respectively; the Rotor-Gene likewise supports six color channels; and Thermo’s QuantStudio systems support from four to as many as 21, depending on the system configuration—the latter thanks to a configurable set of six LEDs and a rotating wheel of excitation and emission filters. That said, most users tend to operate in the one- to three-plex range.

User interface

Another key consideration is system operation. Most qPCR systems, for instance, use either a touchscreen or attached computer to program the system. But systems differ in the number of users and protocols that can be stored, ease of programming, amount of on-board data storage, mechanisms for remote monitoring and data uploading to the cloud and so on—not to mention the user-friendliness of the software itself.

Thermo Fisher’s QuantStudio 3 and QuantStudio 5 systems include wi-fi connectivity, while the 6 Flex, 7 Flex and 12K Flex systems do not, but all QuantStudio systems can upload data to the Thermo Fisher Cloud (a free data-analysis and storage platform). Roche’s LightCycler 96 features a built-in touchscreen, while the LightCycler 2.0, LightCycler 480 and LightCycler 1536 require an external computer. And Agilent Technologies’ touchscreen-controlled SureCycler 8800, like some other platforms, enables remote reaction monitoring.

Price

Price, naturally, is a significant concern, and pricing spans a wide range. Endpoint PCR machines tend to be priced at the lower end of the scale. Roche’s qPCR instruments run from about $25,000 in the United States for the LightCycler 2.0 to nearly $140,000 for the ultra-high-throughput LightCycler 1536. Thermo Fisher’s QuantStudio 3 starts at around $28,000, while the QuantStudio 12K starts at around $90,000 and the dPCR-capable QuantStudio 3D (including all required accessories) costs $44,000.

Performance

And then there are basic performance issues. For instance, how rapidly and accurately the system can change temperature (ramp rate), and the uniformity of heating from sample to sample. Some Peltier blocks can exhibit “edge effects,” in which the temperature is different in peripheral wells than in wells in the block center—a shortcoming that can impact amplification specificity and that is theoretically absent in rotor-based systems. (The QIAGEN Rotor-Gene Q specification indicates a temperature uniformity of +/- 0.02^oC, for instance, compared to +/- 0.4^oC for the QuantStudio 3 and CFX96 Touch.)

Upgradability

Finally, consider upgradability. As mentioned above, many PCR systems include built-in upgradability as it relates to sample-block format, but some systems can be upgraded in other ways, as well. A QuantStudio 6 can be field-upgraded to a QuantStudio 7, for instance—a change that adds support for Thermo Fisher’s TaqMan array cards and boosts multiplexing capabilities from five colors to 21.

Digital PCR instruments are available in two formats of their own. Some, including instruments from Fluidigm and Thermo Fisher Scientific, distribute the reaction into physical microwells etched into the surface of a plate—20,000, in the case of the QuantStudio 3D Digital PCR System. Others, like Bio-Rad’s QX200™ Droplet Digital™ PCR (ddPCR) system and RainDance Technologies’ RainDrop® system discretize the reaction into tens of thousands or even millions of picoliter- or nanoliter-scale droplets, which has the advantage that researchers can increase their assay sensitivity by increasing the number of droplets created or interrogated per reaction. 

Applications

PCR and its variants support a number of applications across the life sciences. Endpoint PCR is used to answer “yes/no” questions. For example: Did I successfully clone my gene fragment? Or: Is a given sequence present in a sample? The technique also can be used to prepare molecules for cloning and libraries for targeted sequencing, introduce site-specific mutations or small insertions (such as a restriction-enzyme cut site) and drive other similar processes.

qPCR and dPCR can be used (when combined with a cDNA-synthesis step) to quantify mRNA and microRNA abundance, probe single-nucleotide polymorphisms, analyze circulating tumor DNA, assess pathogen load and more. Generally speaking, either approach can be used for most applications. But qPCR is more broadly accessible, higher throughput and less expensive, so it is more easily applied to screening—for instance, validating candidate biomarkers first detected via microarrays or next-gen sequencing. Digital PCR, on the other hand, is more precise but lower throughput. qPCR machines also can be used to perform HRM (high-resolution melt) analyses, an alternative assay for genetic polymorphisms.

Both qPCR and dPCR are amenable to multiplexing, meaning researchers can probe more than one sequence in each reaction.

TaqMan qPCR reactions, for instance, can be multiplexed by designing probes for each amplicon using different pairs of fluorophores and fluorescent quenchers, whereas dPCR can be multiplexed by blending multiple colors and dye concentrations.

Researchers can also multiplex endpoint PCR reactions by designing each primer set to produce products of different lengths, which then can be distinguished electrophoretically.

Though all these technologies are mature, new applications are emerging. One new and exciting application for quantitative PCR approaches is in the area of liquid biopsy. Researchers have increasingly embraced the methods to quantify tumor DNA or RNA in blood, urine and other bodily fluids and also to identify actionable mutations, such as genetic variants that can inform a change in treatment. In one recent example, researchers devised a multiplexed ddPCR reaction to quantify and track the evolution of different estrogen-receptor mutations in the circulating tumor DNA of women with advanced breast cancer [1].

Similarly, researchers increasingly are investigating circulating microRNAs (such as those found in extracellular vesicles, for instance) as possible indicators of disease, and they are using quantitative PCR strategies to quantify them (e.g., [2]).

Another particularly popular application is targeted resequencing. Using Illumina’s TruSeq® Amplicon - Cancer Panel, for instance, researchers can amplify 212 segments of 48 cancer-associated genes in one highly multiplexed endpoint reaction. The result is a focused library of specific DNA regions to be sequenced, which helps researchers get more useful data per sequencing run.

Emerging applications include the use of dPCR and qPCR to monitor the progress of genome-editing reactions, to amplify the genetic material from single cells and to track the expression of long noncoding RNAs. 

Table2: PCR Instrumentation*

*Editor’s note: The instruments, methodologies and applications covered in this guide are not intended to represent an all-inclusive view of the offerings in the field. Visit Biocompare's product directory to view the evolving options of PCR products available from leading manufacturers.

Final thoughts

In the 30-plus years since its invention, PCR and its variants have become required tools across the life sciences. The technologies are robust and mature, but that doesn’t mean there isn’t room for improvement, both on the hardware and reagent fronts. Among the features users regularly clamor for are greater sensitivity, higher-efficiency cDNA synthesis, increased multiplexing and greater processivity. New reagents and tools are being explored in all these areas, such as Bio-Rad’s SsoAdvanced reagents, which leverage the Sso7d DNA-binding domain to yield faster amplification, increased inhibitor tolerance, and better overall performance.

Whatever your application, there’s never been a better time for PCR.

The authors thank representatives of Bio-Rad Laboratories, QIAGEN, Roche Molecular Systems and Thermo Fisher Scientific for their assistance in preparing this article.

Reference

[1] Schiavon, G, et al., “Analysis of ESR1 mutation in circulating tumor DNA demonstrates evolution during therapy for metastatic breast cancer,” Sci Transl Med, 7:313ra182, 2015. [PMID:26560360] 

[2] Chevillet, JR, et al., “Quantitative and stoichiometric analysis of the microRNA content of exosomes,” PNAS, 111:14888-93, 2014. [PMID:25267620]