Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Since PCR’s invention in the 1980s by chemist Kary Mullis at the Cetus Corporation, our ability to amplify specific DNA fragments is often taken for granted. Even after 30 years of use, PCR continues to evolve; scientists adapt the basic protocol to address new research questions, with new tools that didn’t exist at the time of PCR’s birth. Beyond the research lab, PCR takes center-stage in genetic identification of people in crime investigations (both real and dramatized) and in genetic diagnoses of diseases in clinics. Take a look at some recent developments in PCR that are speeding researchers’ progress today—Happy Birthday, PCR!
High fidelity enzymes
A secret of PCR’s success is the continuing evolution of the DNA polymerase used to build new DNA strands using a template strand. “Since its invention, improvements in PCR have largely depended on advancements in enzymology,” says Edita Smergeliene, product portfolio manager for gene detection at Thermo Scientific. It’s difficult to imagine the earliest PCR experiments, before temperature cycling was automated by thermal cyclers, and before the discovery of thermostable DNA polymerase. Back then, PCR runs were very hands-on, because researchers had to control sample temperatures manually and add fresh DNA polymerase to each cycle. From the original DNA polymerase derived from E. coli, to the more thermostabile yet error-prone DNA polymerase Taq (derived from Thermus aquaticus), to proofreading DNA polymerases that make fewer mistakes, scientists have tweaked PCR enzymes to meet increasingly specific demands (and free up their time).
Today’s “high fidelity” DNA polymerases feature even more detailed engineering. For example, the Thermo Scientific Phusion High-Fidelity DNA Polymerase is an archaebacterial enzyme with proofreading function alongside a thermostabile DNA binding protein. “This binding protein increases affinity of the polymerase for double-stranded DNA,” says Smergeliene. “The high processivity and robustness of Phusion make the enzyme extremely resistant to PCR inhibitors and enable additional applications not feasible with other more standard polymerases.” EMD Millipore’s high fidelity KOD DNA polymerase is also designed for new PCR applications, such as next-generation sequencing (NGS), developing synthetic biology constructs. “Our PureGenome™ Next Generation Sequencing kits include the benefits of KOD DNA Polymerase to reduce NGS library preparation bias, a common problem in high-throughput, PCR-based sequencing strategies,” says Ayaz Majid, product manager for genomics at EMD Millipore.
New England Biolabs’ recently developed a novel high-fidelity DNA polymerase, the Q5 High-Fidelity DNA Polymerase, that is a fusion with the Sso7d DNA-binding domain. It enables reduced extension times of about 10 seconds per kilobase and fidelity 100-fold higher than Taq. Bio-Rad's high-fidelity, high-processivity PCR reagents also contain the double-stranded DNA-binding protein Sso7d, and are fused to a proofreading polymerase. Their iProof DNA polymerase can amplify long and difficult targets quickly, and is tolerant to PCR inhibitors present in some samples.
Never again will PCR enzymes be “one-size-fits-all.” Kapa Biosystems takes a directed, high-throughput approach for engineering new DNA polymerases for particular PCR applications, such as NGS. “[Our] ability to screen hundreds of millions of unique proteins allows us to identify enzyme variants with dramatically improved functionality compared to native enzymes,” says John Foskett, technical director at Kapa Biosystems, who engineered the new high-fidelity KAPA HiFi DNA Polymerase for amplifying NGS libraries. “It was optimized to reduce PCR-induced bias and improve yield, resulting in more uniform sequencing coverage and increased library complexity.”
One experiment, but many choices
Today’s researchers are blessed with a wealth of enzymes to choose from when planning a PCR experiment, unlike the limited options available at the time of its birth. But how best to winnow down the possibilities?
Most experts agree that it depends mainly on intended applications as well as the nature of your materials. For example, if sequence accuracy of the amplified product is crucial, look for high fidelity enzymes. These are also appropriate for applications requiring amplification of especially long DNA targets. “Some amplifications are challenged by DNA regions with repeat sequences, such as TA repeats, or challenging secondary structures, such as GC-rich repeats,” says Majid. “Here you need robust enzymes with buffer conditions to overcome template challenges.” New England BioLabs’ product manager Fiona Stewart agrees that “it [is] challenging to obtain robust amplification of GC-rich regions and also retain a high level of fidelity in amplification, since high-fidelity polymerases are more susceptible to difficulties in amplification of GC-rich regions.” New Englands Biolab offers the new Q5 High-Fidelity DNA Polymerase to overcome this challenge, and EMD-Millipore offers KOD Xtreme™ DNA Polymerase.
Other situations needing extra care to prevent unwanted mutations might require an enzyme with proofreading activity. If you need high yields of quality DNA for applications such as microarrays or NGS, a high-processivity enzyme with fast extension times may be your choice, such as EMD Millipore’s high-fidelity KOD DNA Polymerase, or the Thermo Scientific Phusion High-Fidelity DNA Polymerase. If the PCR reaction assembly is more convenient at room temperature, such as to use automated robotic systems, or if specificity is particularly challenging, look for a hot-start version of the DNA polymerase.
A source of confusion amongst researchers, notes Stewart, is when to use a conventional versus a high-fidelity DNA polymerase. Typically, high-fidelity enzymes are not needed for routine PCR tasks that might include a quick sequence check or verification of a cloning step. For these jobs, an enzyme lacking the proofreading 3-5' exonuclease activity is likely to be sufficient. However, according to Majid, it’s smart to “look for an enzyme that is highly specific, and sensitive for low-level DNA amplification, such as a NovaTaq™ hot-start polymerase.”
If you find the number of polymerases a bit overwhelming, consider the viewpoint of Kapa Biosystems’ Foskett, who says researchers really only face two choices. “Despite the appearance of a large variety of PCR reagents in the life science market, the vast majority of these reagents are based on either A-family polymerase (Taq), B-family polymerase (proofreading) or a blend of the two enzyme families,” he says. “Any performance differences among PCR reagent suppliers are typically the result of changes to buffer chemistry or enzyme concentration, with very little innovation at the protein level.”
Recent innovations
Digital PCR. One of the most exciting innovations in PCR today is digital PCR, which allows you to count the absolute number of DNA molecules in a sample. Systems available for this include Life Technologies’ QuantStudio™ 3D Digital PCR System, Bio-Rad Laboratories’ QX100 droplet digital PCR (ddPCR) system, Fluidigm’s qdPCR 37K™ IFC system, and RainDance Technologies’ RainDrop Digital PCR System. Most systems work by partitioning the DNA sample into thousands or millions of partitions or droplets that are so small they only contain one or zero molecules of DNA, which are then counted. According to Rachel Scott, amplification instruments business unit marketing manager in the gene expression division at Bio-Rad Laboratories, “due to the increased sensitivity and precision of digital PCR, ddPCR is uniquely suited for absolute quantitation, copy number variation and rare event detection.” Angelique Habis, senior market development manager of genetic analysis for Life Technologies, notes additional applications of digital PCR, including rare target detection from cancer samples, pathogen detection, absolute quantitation of viral load, detection in genetically modified organisms, quality control of NGS libraries and the generation of reference standards.
Direct PCR. Another innovation is “direct PCR,” in which PCR is performed directly on a collected sample such as tissue or blood—without the usual preparatory DNA-purification step. Today’s chemistries are making this possible. “To be able to amplify in such conditions, the polymerase has to be quite resistant to reaction inhibitors, such as constituents of blood,” says Smergeliene. “DNA polymerases with increased processivity, such as Phusion or Phire enzymes, have this additional beneficial feature and are at the core of Thermo Scientific Direct PCR kits.”
Improvements in thermal cycler design. Other recent PCR advances include improvements in instrumentation that accomplish several goals, including reduced sample volumes (which save precious, rare sample materials), cost savings on reagents and reduced environmental impact via decreased waste. In addition, improved thermocycler designs enhance reaction efficiency and save lab space. For example, using smaller reaction volumes (down to 5 µl) enables Thermo Fisher Piko Thermal Cyclers to use plates that are one-quarter of the standard size. The company also offers ultra-thin-wall plates and tubes for better thermal transfer and reduced waste. These “give the user time, reagent, space and energy savings,” says Kari Kataja, product manager for PCR/qPCR instruments, sample preparation and analysis at Thermo Fisher Scientific. “The small format and instrument size enable us to accommodate these low-noise and low-weight instruments on the benchtop for personalized use.” Other thermal cycler features are improving the accuracy and reproducibility of PCR, such as Eppendorf’s “thermal blocks with high temperature accuracy and homogeneity, and evaporation-reducing lids,” says Kay Koerner, global senior product manager at Eppendorf.
Looking ahead
Just as it was impossible to chart the course of PCR three decades ago, it’s unclear where the technique is heading next. “No one would have predicted 30 years ago that such a simple concept of copying DNA would transform biology and human health,” says Foskett. Today, new applications of this simple concept continue to spark scientific imaginations. “We are excited about single-cell genomic analysis, enabled by novel high sensitivity, low-bias whole genome amplification methods, for applications such as pre-implantation genetic screening,” he says. Indeed, we’ve likely observed only the beginning of PCR’s power. After all, at 30 years old, PCR is in its prime.