by Caitlin Smith
It really is science, right—not magic? Finding the best conditions for your PCR protocols can be frustrating and time-consuming, but it is becoming less so. Here we look at some tools to help you arrive more quickly at your best-results destination. Tom Evans, the division head for DNA enzymes and polymerases at New England BioLabs, says that “optimizing the PCR workflow includes improvements in the sample prep time, the reaction time, reaction sensitivity, reproducibility, and ease of use.” The technologies here are some examples of current tools that accomplish this.
Extraction-free PCR and optimizing mixes
The chemical building blocks and solutions that make your PCR reactions happen are crucial for routine success. How do you know which ones work best, when there are so many to try? These and similarly innovative tools may help.
Evans notes that within the PCR workflow, a significant amount of time is spent preparing the DNA. “Eliminating this step results in significant time and cost savings,” he says. “New England Biolabs provides a number of PCR products that permit ‘extraction-free’ PCR. Hemo KlenTaq™ DNA polymerase and the Phusion® Blood Direct PCR Kit are tolerant of greater than 20% of the PCR reaction volume being whole blood. Similarly, the Phire® Animal Tissue Direct PCR Kit and Phire® Plant Direct PCR Kit permit PCR directly from a variety of animal and plant tissues.” (Phusion® and Phire® products are manufactured by Finnzymes Oy and distributed by New England Biolabs.) The condition of your DNA substrate can also affect PCR optimization, according to Evans. “We supply a unique product, the PreCR™ Repair Mix, that can accurately repair damaged DNA templates,” he says. “Innovative products such as this, as well as the ‘extraction-free’ PCR products permit the limits of PCR to be continually challenged.”
PCR optimization can be time-consuming, but Epicentre’s FailSafe™ PCR PreMix Selection Kit can help you optimize the solutions best-suited for each template-primer pair in less time. Their kit contains 12 PreMixes that cover a range of PCR conditions, such that each PreMix represents a unique set of PCR conditions best suited for a given type of sequence. At least one PreMix will give optimal conditions for the template and primers that you test, as guaranteed by Epicentre. You can then order the one PreMix that gives the best results for your subsequent PCR. “Our FailSafe™ PCR system is still very popular for optimizing PCR of both routine and challenging templates,” says Ken Doyle, director of marketing at Epicentre Biotechnologies. “The main advantages of the FailSafe™ system are that it works extremely well for templates with high GC content (greater than 80%) and is also suitable for multiplex PCR. The FailSafe™ system also gives longer PCR amplicons (over 20 kb and even ~30 kb with some templates) compared to competitive kits.”
Alternatively, Qiagen maintains that time need not be spent searching for the optimal PCR mix, because their PCR buffers are designed to work for a range of PCR reactions and templates. “Thanks to the unique buffer composition provided in Qiagen PCR Kits, an optimization [for] PCR parameters is not necessary,” says Svea Lübcke, associate director of marketing PR at Qiagen. “Our kits include the novel reagent Q-Solution, which changes the dynamics of the PCR reaction to increase sensitivity when amplifying GC-rich DNA and targets with strong secondary structures. Our innovative PCR buffer formulations contain a balanced combination of K+ and NH4+, which strongly increase primer annealing specificity.”
Lübcke says that PCR success largely depends on maintaining a high ratio of specific to nonspecific annealing of the primer molecules. “Annealing is primarily influenced by the components of the PCR buffer (in particular the cations) and annealing temperature,” she says. “Qiagen has found that the balanced combination of K+ and NH4+ used in all Qiagen PCR buffer formulations can strongly increase primer annealing specificity over a broad range of annealing temperatures. The improved specificity is caused by ammonium ions destabilizing the weak hydrogen bonds at mismatched bases. The use of these buffers also often eliminates the need for optimization of Mg2+ concentration or annealing temperature for different primer-template systems and maintains high primer annealing specificity in each PCR cycle.”
Time-saving DNA polymerases
Even your choice of enzyme can help save time. For example, Evans says that Phusion® and Phire® DNA polymerases save time because of their fast extension times. “In addition, these polymerases can be used in a two-step PCR protocol, instead of the more standard three-step protocol,” he says. “These factors can result in 2-3 times faster reaction times, as compared to other typical PCR enzymes and protocols.” New England BioLabs also offers help with hot-start protocols with their Affibody® inactivation technology, which “results in a ‘zero-time’ reactivation of both Phusion® and Phire® Hot Start DNA polymerases,” says Evans. “No additional heating at high temperature is needed to activate the DNA polymerase for PCR. This saves not only time, but also reduces the possibility that the extended high-temperature incubation may damage the DNA.”
Bio-Rad also offers new high-performance enzymes designed to enhance speed, yield, and sensitivity. “PCR enzyme development is an exciting area that is impacting our ability to do faster PCR reactions with higher yields,” says Pete Skirpstunas, product manager in the gene expression division at Bio-Rad. “In particular, fusion enzymes such as the Bio-Rad iProof DNA Polymerase offer more robust activity and higher speeds.”
Optimizing thermal gradients
Using a thermal gradient in your cycler can help determine the optimal annealing temperature for your primers, maximizing their specificity. “Using a thermal gradient will allow you to find the optimal annealing temperature in a single experiment, saving you time and money on reagents,” says Skirpstunas. “Bio-Rad thermal cyclers use a dynamic ramping gradient technology. All rows (or columns) reach their desired temperature in unison. This results in a more direct transfer of your protocol from optimization to experiments.” Skirpstunas says you can quickly optimize your protocol speed by using the Bio-Rad C1000 thermal cycler’s protocol autowriter, which “will determine the best protocol based on your input of amplicon length, annealing temperature and desired speed.”
Eppendorf offers a thermal gradient block on all their Mastercycler® pro thermal cyclers. “This is still one of the most critical applications for PCR optimization,” says Beverly Cutson, product manager for PCR and detection at Eppendorf. “All of our gradient thermoblocks use our SteadySlope® technology; this ensures that the ramp rates remain constant during both the gradient optimization and the actual experimental run. Ramp rates can affect the outcome of the reaction. By keeping the ramp rates the same we remove this variable, thereby simplifying the optimization process. In addition, the vapo.protect™ technology ensures optimization by decreasing the evaporation drastically. Evaporation during the PCR reaction can lead to failure.”
Optimizing PCR reactions will continue to get easier as technology improves. Evans notes that “a universal buffer-polymerase combination that works well on low GC content DNA, stretches of nucleotide repeats, and avoids ‘stuttering,’ is a significant challenge, but would be a boon to the scientific community.”