It is impossible to overstate the importance of the polymerase chain reaction (PCR). The Nobel Prize-winning DNA amplification technology drives modern genomics, forensics, medical diagnostics, and more. Still, the technique is not necessarily easy to do or without complications. Due to its sensitivity, PCR is highly prone to contamination and off-target effects. When designing a working polymerase chain reaction, primer design, reaction conditions, and enzyme selection must all be considered. This complexity is compounded in multiplex PCR, in which multiple targets (usually between two and five) are detected simultaneously in the same tube. Applications include gene expression analysis, SNP genotyping, forensics (e.g., STR typing), and pathogen detection.
Multiplex PCR is economical—with fewer reactions, there is less reagent consumption. It is frequently used when dealing with precious samples (e.g., clinical samples), or to enhance throughput (with fewer microtiter wells per sample there are more samples per plate).
Challenges of PCR multiplexing
A traditional PCR reaction contains the template DNA, two primers flanking the amplification site, an enzyme, and buffer. The resulting amplicons are generally detected by gel electrophoresis. A quantitative, real-time PCR reaction typically includes all of that plus a probe that can be detected fluorescently as the reaction runs, with no gel required.
Multiplexing such a reaction amplifies the design challenges. If one target requires three primers, none of which should be able to bind elsewhere in the template DNA or to each other, then two targets require six, three require nine, and so on. Each amplicon must be either a different size (for gels) or labeled with a fluorescent dye that is spectrally distinct from the others in the reaction. Complicating matters is that different targets in the reaction can compete with each other for resources such that highly abundant templates are detected, and less abundant ones fade into the background.
As a result, says Jonathan Wang, Senior Scientist at Life Technologies, multiplex PCR is to some extent a niche application. “Most people think it’s too difficult.”
“There are a lot of customers interested in multiplex PCR, but because of the challenges, they have stayed away,” adds Laura Mason, Product Manager at Agilent Technologies.
Fortunately, for those who must use the technique, there are plenty of tools and tips available to help.
Nicole Nichols, Senior Application and Development Scientist at New England Biolabs, offers the following five basic rules for researchers designing multiplex PCR reactions.
Primers for PCR
Rule 1A: Primer design
The first rule focuses on primer design. “There’s really no substitute,” Nichols says. “Good attention to detail at the beginning will save a lot of optimization time at the end.”
She recommends using slightly longer primers than usual (24–35 bases), with slightly higher melting temperatures (65 °C or higher) and a GC content of 50–60%. Naturally, that is in addition to all of the other standard PCR primer design rules: maintain a similar melting temperature across all primers; avoid complementary sequences and runs of three or more Gs or Cs at the 3’ end (these can cause mispriming reactions); and validate, validate, validate.
For real-time multiplexed PCR—which typically is performed using a 5’-nuclease-based assay, for instance using Life Technologies’ TaqMan® or Integrated DNA (IDT) Technologies’ PrimeTime® probes (intercalating dyes such as SYBR Green cannot be used in multiplex reactions)—Keith Cockrum, a Genomic Development Specialists Manager at Bio-Rad, recommends designing those probes such that their melting temperature is 7–8 °C higher than the amplification primers themselves. “We need the probe to anneal first,” he explains. Otherwise, the sequence could amplify without the probe, an undetectable reaction he calls “endogenous PCR.” “It’s the PCR event we want—it’s not nonspecific—we’re just not getting any light from it,” he explains.
Rule 1B: Primer validation
Validation is a multistep but essential process, says Cockrum. First, he advises, take every primer pair and test it individually on the template (control samples, such as Agilent’s QPCR Reference Total RNA, can provide some standardization here). Make sure it produces a single product, for instance using a SYBR Green melt curve. Run a temperature gradient to define the window over which all sets work, Nichols adds, and pick running temperatures near the higher end of that window. If one primer pair does not work with the others, she says, trash it and start again. “You’re better off throwing [it] away and redesigning rather than trying to optimize.”
Next, says Cockrum, test your probe oligos (if doing real-time PCR) in single-plex reactions over a range of dilutions to define the dynamic range of each one. Finally, put everything together to make sure specificity, sensitivity, dynamic range, and so on remain the same. “If you demonstrate all those things, then you have an amazing, awesome multiplexed PCR assay,” Cockrum says.
Rule 2: Primer concentration
Nichols’ second rule is to keep the concentration of each individual primer lower than in single-plex reactions, because “the total primer concentration in the reaction can get too high.” Instead of 0.2 μM per primer in a traditional reaction, try 0.15 μM per primer (though the range is 0.05 μM to 0.4 μM).
Rules 3, 4, and 5: Optimize amounts of dNTPs, MgCl2, polymerase, and salts
Next, Nichols says, tweak your reaction conditions. With so many reactions running at once, “you don’t want the reaction to run out of the components.” Multiplex PCR reactions typically contain extra dNTPs and magnesium—rule no. 3—and polymerase (rule no. 4). Nichols recommends about 0.3 mM dNTP (vs 0.2 mM standard) and 2.5 mM Mg2+ (vs 1.2 to 2 mM), and 5 units of polymerase per 50-μL reaction, as opposed to 1.25 units normally.
Finally, she says, try salt (rule no. 5). “When optimizing, increasing the salt can also help, particularly with specificity.”
Users can approximate rules 3–5 using conventional PCR master mixes (which contain buffer, salts, dNTPs, Mg, and polymerase) at 1.25× or 1.5×, Nichols says. Alternatively, they can opt for multiplex-optimized master mixes, such as New England Biolabs’ Multiplex PCR 5× Master Mix, Bio-Rad’s iQ™ Multiplex Powermix, or Agilent Technologies’ Brilliant Multiplex QPCR Master Mix.
The Brilliant Multiplex QPCR Master Mix, for instance, is specifically formulated to address competition between different reactions in the same tube, says Agilent’s Mason. If one target sequence is present 1000 times, and another is present 10, the former could effectively quench the latter. “Our multiplex kit is formulated to allow accurate quantification of both low- and high-abundance targets in the same tube, thus overcoming the bias,” Mason says.
Maximum number of targets/reaction
Generally speaking, explains Cockrum, the maximum number of reactions that can be detected simultaneously in one tube is five—a constraint imposed by both primer design considerations and the fact that multiplex real-time PCR reactions are monitored using real-time PCR instruments, which generally can detect no more than four or five color channels, one of which may be reserved as a reference channel.
Though endpoint (i.e., gel-based) PCR does not suffer from that particular limitation, the challenge of getting multiple primer sets to work together remains, meaning they still max out at about five-plex reactions. “Designing anything more than that becomes inordinately challenging,” Cockrum says.
Dye sets for real-time PCR systems
Real-time instruments capable of multiplex PCR include Life Technologies’ QuantStudio™ 12K Flex Real-Time PCR System and Agilent’s Mx30005P. Bio-Rad’s CFX96 Touch™ Real-Time PCR Detection System is a five-channel instrument with separate excitation sources and detectors for each channel, Cockrum says. In a typical configuration, channel 1 is assigned to FAM, channel 2 to HEX or VIC, channel 3 to Texas Red or ROX, channel 4 to Cy5, and channel 5 to Quasar 705. The most popular choices, though, are HEX, FAM, and VIC, with FAM/HEX and FAM/VIC being the two most common combinations, Cockrum says. Life Technologies, which produces VIC, recently released two new dyes to early access customers, ABY and JUN, as well as a new passive reference dye, Mustang Purple.
Cockrum recommends matching dye intensity with template abundance, such that the brightest dye (usually FAM) is paired with the rarest template, and the dimmest dye (e.g., Cy5 or Quasar 705) is paired with the most abundant housekeeping genes.
More multiplex: detection of 10 or more targets
Of course, primer design is the biggest hurdle to multiplexing, but once this hurdle is overcome it is possible to go higher. Though it does not simplify primer-design difficulties, Agilent’s MassCode technology does allow multiplexing well beyond real-time instruments, and Mason says the company recommends this technology for those interested in screening 10 or more targets simultaneously, for instance, in pathogen detection.
MassCode oligos are not tagged with fluorophores but with photocleavable chemical tags of varying molecular weight that can be detected in a mass spectrometer following amplification and exposure to UV light. In 2011, company scientists demonstrated a 14-plex panel, which they used to detect and serotype various strains and serovars of Salmonella.1
Primer design software
So how does one design such compatible primer sets? In a word, software. There are a variety of free calculators available to help with this process, such as Integrated DNA Technologies’ RealTime PCR calculator. Nichols prefers Primer3 (MIT, Cambridge, MA), which she supplements by running potential primer sequences against the UCSC Genome Browser to guard against off-target priming.
Alternatively, there are commercial options, such as Premier Biosoft’s Beacon Designer (also available from Bio-Rad). According to Abhimanyu Holkar, Genomics Product Manager at Premier Biosoft, the software aids in multiplex assay design by using genome-wide BLAST searches to avoid repetitive sequences, structure prediction tools to avoid highly folded regions and pairwise testing to remove likely primer-dimers. From millions of potential combinations, he says, the user is presented with a small set of potential pairs matching the input criteria, which can then be tested in the lab.
Before doing that, though, be sure multiplexing actually makes sense. Many may feel that the benefits of multiplexing—sample usage, cost, and throughput—are offset by the time required for optimization and validation. That is especially true given the availability of relatively low-cost, predesigned single-plex assays from Life Technologies (TaqMan), Bio-Rad (PrimePCR™ SYBR Green-based assays), and others.
“Once you get multiplex working, it’s working,” says Cockrum. “But if you [design assays] only once a year, it will be complicated each time you do it.” Still, if one must multiplex, tools exist to make the process as painless as possible.
1. Richmond, G.R.; Khine, H. et al. MassCode liquid arrays as a tool for multiplexed high-throughput genetic profiling.PLoS ONE 2011, 6(4):e18967.
The image at the top of the page is from Bio-Rad's qPCR Assay Design and Optimization.