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.
The Search Enzymes was invented just about 30 years ago, with the thermostable Thermus aquaticus DNA polymerase (“Taq”) and the automated thermocycler quickly following suit. Life sciences research has never been the same.
The patent on Taq has long expired, most vendors now offer one or more Taq-based enzymes for routine PCR, and Taq is still the standard against which other PCR polymerases are measured. So, is there any reason to use anything else?
It depends. For routine amplification applications like genotyping or checking for the presence of an insert, “you need something robust, consistent and at a good price. There are some good options out there” in Taq-based polymerases, says Karen Martin, senior product manager at Clontech. “
For this, you don’t really need an expensive polymerase that has a lot of bells and whistles.”
But not every application is routine, and Taq has a reputation for replication errors. For cloning or next-generation sequencing, where accuracy is required, an enzyme with proofreading abilities—generally termed “high-fidelity”—is in order, she notes.
Crank up the hi-fi
“Typically DNA polymerases are stratified into high-fidelity and those for routine PCR,” says Breton Hornblower, DNA amplification product marketing manager for New England Biolabs (NEB).
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Such stratification becomes obvious when searching for enzymes with high processivity or the ability to handle difficult or “dirty” templates—these are often (but by no means always) one and the same enzyme or variations on a theme.
High-fidelity comes in a variety of formulations, and even the meaning of the term can vary among manufacturers (and among a single manufacturer’s offerings). For example, NEB’s OneTaq® “is a blend of Taq and a proofreading enzyme that results in increased robustness and also increased fidelity vs. standard Taq,” Hornblower says. NEB’s website lists high-fidelity PCR as an application, but Hornblower says OneTaq is actually the company’s recommendation for routine PCR. For high-fidelity applications, he says, the company’s choice is its Q5® High-Fidelity DNA Polymerase, which offers “more than 100 times the fidelity of Taq.”
Others use different enzymes altogether. The online description for Life Technologies’ Platinum® Pfx DNA Polymerase, for instance, says “high fidelity is provided by a proprietary enzyme preparation containing recombinant DNA polymerase from Thermococcus species KOD [kodakaraensis] with proofreading (3´→5´ exonuclease) activity.” High-fidelity enzymes from various Pyrococcus species, also with 3´→5´ proofreading ability, have been commercialized by several vendors as Pfu DNA polymerase and under trade names such as Deep Vent (from NEB). Similarly, Bio-Rad Laboratories says its iProof™ high-fidelity polymerases comprise “a unique Pyrococcus-like proofreading enzyme” fused to a double-stranded DNA-binding protein called Sso7d.
Hang on
Several other vendors have licensed the right to incorporate Sso7d—the nonspecific DNA-binding domain of the hyperthermophilic archaeon Sulfolobus solfataricus—into their polymerase formulations, as well. These products include NEB’s Q5 product and Thermo Fisher Scientific’s Phusion™ DNA polymerases. Similarly, Agilent Technologies’ PfuUltra II Fusion HS DNA Polymerase is composed of a different, proprietary, double-stranded DNA-binding protein fused to a Pfu-based DNA polymerase.
“If you’re looking at a crude lysate or sample that has some inhibitors that were carried over, and it might be GC-rich, and/or you’re looking at long PCR, then we would recommend the fusion-style enzyme due to the performance of that enzyme,” notes Paul Streng, senior product manager for real-time PCR Reagents at Bio-Rad Laboratories. The domain “acts like a clamp and it binds itself to the template a little bit tighter.” This, in turn, enables the enzyme to deal with certain more difficult amplification reactions, because it is less prone to fall off in a heavy secondary structure or in the presence of PCR inhibitors.
Other high-fidelity formulations are sold as solutions for researchers looking for processivity or the ability to deal with difficult samples, as well. For example, Clontech’s Takara PrimeSTAR® GXL “has a good level of fidelity, but it is also very accommodating,” says Martin. “High GC-rich templates are no problem, [and] long targets (30 kb) are okay. It’s got a very strong processivity, so it can handle both situations.” And, she notes, too much template (with which some polymerases can inhibit the reaction) is not a problem.
Because most high-fidelity solutions use a 3’ → 5’ exonuclease activity for proofreading, the resulting PCR products will likely have blunt ends, so plan accordingly for downstream applications such as TA cloning.
Special situations
It’s tough to ferret out genuine differences from marketing hype, and this article makes no pretense at being able to do so. But some products are sold specifically for ultra-high fidelity, long amplifications, speed, or other nonstandard PCR applications.
Among these are some version of direct PCR—from Clontech and Thermo Scientific, for example—in which samples like plant tissue or mouse ear plugs can be amplified without prior extraction and purification. And NEB’s Hemo KlenTaq® is sold to amplify nucleic acids directly from whole blood by using a mutated, truncated version of Taq that is resistant to inhibitors. Be aware that as a tradeoff, 3’ → 5’ proofreading often is compromised in such polymerases.
Some polymerases, says Hornblower, are designed to incorporate modified or labeled nucleotides. Agilent’s PfuTurbo Cx Hotstart enzyme “can read through uracils, making it ideal for doing DNA methylation studies,” notes associate marketing director Surekha Karudapuram. And the company’s PicoMaxx PCR system is designed to detect low-copy-number targets without extensive purification and subsequent DNA loss. “These are examples of niche, specialized applications where you can’t use just any old enzyme,” Karudapuram says.
The heat is on
Of course, there’s more to PCR than polymerase. Formulation matters, too. Many manufacturers offer different buffer or master-mix formulations for different applications, such as dealing with GC-rich environments, notes Streng.
Some of those may include a hot-start option that prevents polymerase and exonuclease activities from taking place before the reactions are heated. Different formulations work in different ways (generally involving an antibody, aptamer or chemical blocker attached to the polymerase) and with different kinetics, but the overall idea is the same: “It’s convenient not to [have to] set up everything on ice, but more importantly it prevents nonspecific amplification prior to the start of PCR,” said Karudapuram.
If you’re confused, you’re not alone. Many vendors—especially those with many different DNA polymerases in their portfolio—have selection guides on their websites to help researches through the decision process. (See also a recent Biocompare Bench Tip article from Bioline offering advice, as well.)
Some, like Clontech, let researchers try the enzymes in their own hands by offering free samples. (Note that cycling parameters—especially annealing temperatures—may differ between standard and high-fidelity PCR reactions.)
Streng goes a step further, encouraging side-by-side comparisons. If the results are the same, “then it’s a price decision. But if there’s a difference, that should dictate which to choose.”