In an effort to discover all factors constraining the speed of polymerases in the PCR reaction, a University of Utah group headed by Carl Wittwer has developed a fluorescent probe to monitor polymerase activity.
In a trio of recent studies using this probe, the group has begun to define the various rate-limiting factors in the extension phase of PCR. Temperature and base content of a strand of template were shown to influence speed. Monovalent cations and secondary structure slowed down rates, and addition of just the right amount of DMSO could speed them up.
Characterizing polymerases in this manner, and discovering ways to drive them to maximum speeds, may be useful to high-throughput PCR and reactions where the ideal speed is fractions of seconds.
In an interview with PCR Insider, Jesse Montgomery, first author on the lab's recent study published in The Journal of Molecular Diagnostics said that, surprisingly, this type of work on polymerases has never been done comprehensively. The lab's fluorescence-based technique also has advantages over the gold standard Kornberg assay, which uses radiolabeled dNTPs. Moreover, it was developed to work on a real-time PCR platform commonly available in labs, the LightCycler 480 from Roche.
"There are several different polymerase assays out there," Montgomery said, "but none really see the light of day. We know that the hiccups are complicated instrumentation and chemistry. So we knew we needed to adapt this for a more common platform ... a real-time PCR instrument."
"What we're really trying to get at is to understand how fast PCR can be performed. We've made amazing progress; we can get full 35-cycle amplification, to plateau, in less than 15 seconds," Montgomery said — a feat that Wittwer highlighted in various conference presentations last year.
Montgomery added, however, that "there's obviously a lot that we don't understand about how fast this process goes, so we really are trying to separate each reaction — annealing, denaturation, and extension — and perform these experiments where we can isolate each reaction and measure the kinetics."
The fluorescence method uses a stopped-flow technique originally described by the Utah group in Analytical Biochemistry. It employs an intercalating dye to determine extension rates, measured as nucleotides per second per molecule of polymerase. That study compared activities of 15 commercially available polymerases, and showed "large differences between different types of polymerases, and that they perform quite differently in different buffers," Montgomery said.
To follow this up, the Wittwer lab published a second study in Clinical Chemistry to evaluate the most optimal chemistry conditions for polymerases.
"We looked at common PCR additives, and found some surprising results, including a few things that are against common convention," Montgomery said. For instance, monovalent cations, such as amonium sulfate and potassium chloride, are commonly used in PCR buffers. These "really strongly" inhibit polymerase activity. "At a typical concentration of 25 to 50 millimolar, you're losing 50 to 60 percent of your activity," Montgomery said. "We're finding that just about everything you put in PCR buffers affects polymerase activity."
The group dialed in on optimal conditions and made recommendations in the Clinical Chemistry paper, but then wanted to look at the effect of the template itself and the thermocycling conditions typically used in PCR.
Thus, in the most recent paper they looked at a range of different templates with different GC contents, for example, and found "a pretty drastic effect" of a five-fold difference in rate between a template with 20 percent versus 60 percent GC content.
"This is really important. When we're trying to push PCR to its absolute limit, we have to start considering factors that we've always taken for granted," Montgomery said. "Do we need to adjust our thermocycling conditions to match each individual template? Our findings show, yes we do, we need to consider the GC content. We found [that] Taq varieties of polymerase … incorportate G's and C's much quicker than A's and T's, but Pfu polymerase is actually the opposite."
In the paper, using a thermostable, deletion-mutant polymerase at a temperature of 75°C, the authors showed that A's were incorporated at a rate of 81 per second, C's and G's at 150 and 214 per second respectively, and T's at a rate of 46 per second, according to the study. Meanwhile, 7-deaza-G – an analog with a lower melting temperature than G when paired with C – was handled at 120 per second, while the rate for U's was half that of T's and did not increase with increasing concentration.
Knowing these rates, Montgomery and his co-authors wanted to see if they could predict extension rates for a random piece of template with known base composition. "It was really close in a lot of cases, for two it was right on, so then the question was, is there something that is keeping it from being as fast as it possibly can be based on the nucleotide sequence?"
Montgomery said he hypothesized that secondary structure could be slowing down the reaction. DMSO has been suggested to relieve secondary structure in PCR, and his experiments with this "really brought up extension rates much closer to prediction, but for the ones that were already matched to prediction, it didn't really change the extension rates at all," Montgomery said.
"We came to a realization that the speed of polymerase extension is strongly influenced, probably determined, by nucleotide composition, and inhibited by secondary structures," he said. "To really reach a maximum possible extension, it's good practice to add 5 to 7 percent DMSO to relieve secondary structure."
"When we're trying to get as fast as we possibly can, down to PCR in a matter of seconds, we need to pay attention to these parameters that don't really come into play when we're talking about 15 or 20 minute PCRs," he added.
Montgomery said the group has been pursuing a patent for the fluorescence polymerase assay, including the stop-flow and real-time PCR platform-suitable assays. They have also seen interest from companies wanting to replace the radioactive Kornberg assay in their quality control, as well as companies asking the lab to measure the speeds of their polymerases, to answer the basic question "How fast is it?," Montgomery said. The assay could also be useful in characterizing polymerases from different organisms as well as for optimizing the chemistry in master mixes, he added.