Making it Possible to Amplify More Accurately, Even with Problematic Sequences
To get enough DNA for processing, such as sequencing the chain of nucleotides, researchers turn to the polymerase chain reaction (PCR). A series of reagents drive this process, and that includes polymerase, buffers, and so on. To make it easier for scientists to use this technique, vendors make kits of PCR reagents. As PCR moves forward, taking on more difficult stretches of DNA to amplify, the reagents must advance in lockstep. In fact, every advance in PCR platforms demands concomitant improvements in the PCR reagents to get the most from the advancing technology.
“Robustness is a recurring theme with PCR reagents, and it has been for many years,” says Fiona Stewart, Ph.D., PCR product manager at New England Biolabs (Ipswich, MA). For the most part, robustness describes reliability and consistency of the reagents. For example, forensic labs apply PCR to many materials from crime scenes to analyze DNA samples, and those processes must produce reliable results. In these cases, lives literally hang in the balance, in part determined by the consistency of the PCR reagents.
Pushing ahead with the platform
“Trends in PCR reagents tend to go hand in hand with advances in instrumentation,” says Rod Pennington, Ph.D. senior research scientist at Promega (Madison, WI). “For instance, we see a trend toward reagents containing brighter, less-inhibitory (double-stranded) DNA binding dyes. This enables users to utilize the full HRM (high-resolution melt) potential of current thermocyclers that feature finer temperature control.” He adds that today’s PCR platforms often provide faster cycling with high throughput, and reagents must be designed for that.
Today’s reagents can also help scientists find specific targets in samples. As Pennington says, “In some cases, researchers need reagents that are custom produced and/or preloaded with primers and probes for detection of specific targets.”
The need for speed
At the real-time PCR research and diagnostic core facility of the School of Veterinary Medicine at the University of California, Davis, director Emir Hodzic, D.V.M., Ph.D., often gets requests for fast turnarounds on samples. “Sometimes our clients ask for results within a couple of hours,” he says. “To go that fast, we need to change the thermal cycler’s block. That’s an obstacle to speed, and then you need to do some optimization.”
Trisha Dowling, director of product management for PCR at Life Technologies (Carlsbad, CA), points out that “there are differences in thermal cyclers that are optimized for faster PCR reactions.” For example, she says, “Our Veriti thermal cycler is optimized to run both standard and fast PCR protocols at a range of volumes. Our GeneAmp PCR System 9700 offers multiple block choices of different alloys for faster sample ramp rates.” To run the process faster, the thermal-cycler block needs to accommodate faster temperature changes and the enzyme must also work at the new rate. By using a smaller volume, the sample’s heat can be changed faster and the enzymatic reaction works faster across the sample, too. “All those components must work together,” she says.
Another key factor to faster reactions is ease of setup and programming. As Dowling says, “Our Veriti thermal cycler has an intuitive touchscreen that makes it very easy to input and quickly start your PCR protocol, or to easily access one of the many pre-programmed methods.”
Scientists also expect today’s PCR reagents to work more or less upon delivery. “They want good results with minimal optimization, regardless of the template,” says Stewart.
In the past, different DNA templates required different polymerases for amplification. “Now, there’s no reason to have a bunch of polymerases in your freezer,” PCR Reagents Making it possible to amplify more accurately, even with problematic sequences June 2012 Lab Manager 59 says Stewart. “In fact, researchers want to see good results for all of their PCR reactions using a single polymerase and—as often as possible— a single buffer.”
Pennington also notes the trend toward simplification of the PCR workflow. He says, “For instance, inhibitorresistant PCR enzymes and buffer formulations can allow the user to avoid certain time-consuming steps in their sample preparation.”
Charles Nicolet, Ph.D. director of sequencing technology at the University of Southern California’s Epigenome Center Data Production Facility, says, “We have been using a 2X master mix from Kapa Biosystems for our PCR. It is stable, easy to use, and robust. It amplifies regions with little to no apparent bias. Technically we would not change anything.” He adds, “I suppose economics are always an issue. The Kapa enzyme is very competitively priced, but if anything could be changed, then making it cheaper is always appreciated.” As researchers apply PCR to even more research questions and use it increasingly as part of the sample preparation for other processes, such as next-generation sequencing, price could turn into an even bigger issue.
As expected, speed
For most any trend related to lab work, people want things to work faster, and PCR is no exception. In fact, researchers tend to want more and faster. With PCR reagents, that means researchers want the process to run faster and work with, as Stewart says, “more difficult amplicons.” For example, she says, “GCrich amplicons have always been difficult to amplify, but we’re making significant headway there.”
On the speed side, the reagents really stretch the possibilities. “We’re pushing the limits of speed,” Stewart says
In addition, researchers want to set up their PCR reactions at room temperature. “So they want to use hot-start reagents, whose polymerase is inhibited at lower temperatures and activated during PCR cycling,” Stewart says.
Fidelity of amplification—getting the nucleotides right—also keeps increasing with advances in PCR reagents. In some cases, researchers simply use PCR for a yes-no assay—one that just looks to see if a sequence exists in a sample. In such cases, there’s no reason to pay the higher price for increased fidelity. On the other hand, if a researcher uses PCR to make libraries for next-generation sequencing, higher fidelity from the PCR impacts the accuracy of the end result from the sequencing. In those cases, a researcher wants as much fidelity as possible from the PCR reagents
Some applications of PCR raise the accuracy bar higher than ever. As Pennington says, “With PCR being increasingly popular in forensics and environmental testing, there is a need for reagents that function well in the presence of inhibitors and with less-than-pristine samples in general.”
In the future, PCR will continue to expand to new areas, with the reagents driving that in part. “Highthroughput reagents are particularly well-suited for assays run repeatedly on large numbers of samples, as might be seen in a diagnostic setting,” says Pennington. He also notes the potential of digital PCR, which provides more precision than traditional PCR. He says, “Digital PCR opens up new and better options for quantification of low-copy targets.” He adds, “Improved reagents and technologies for HRM allow this technique to be applied to analyses of difficult SNPs (single nucleotide polymorphisms) that weren’t amenable to HRM analysis before the advent of these improvements.”
So the advances in PCR reagents push this technology into more applications. The more sophisticated reagents also make it easier for scientists to use this technology, all while getting more accurate answers in less time.
For additional resources on PCR reagents, including useful articles and a list of manufacturers, visit http://www.labmanager.com/reagents