DNA polymerase is an essential component for PCR due to its key role in synthesizing new DNA strands. Consequently, understanding the characteristics of this enzyme and the subsequent development of advanced DNA polymerases is critical for adapting the power of PCR for a wide range of biological applications. Since the use of Taq DNA polymerase in early PCR protocols, significant improvements have been made specifically in the specificity, thermostability, fidelity, and processivity of PCR enzymes. These properties of DNA polymerases have been modulated in combination to enhance PCR as described in the sections below.
Nonspecific amplification is one of the major hurdles in PCR since it can drastically impact yield and sensitivity of target amplification, thereby compromising interpretation of results and the success of downstream applications. DNA polymerases often extend misprimed targets and primer-dimers, which are common sources of nonspecific amplification. One way to reduce nonspecific amplification is to set up PCR on ice. This helps keep the activity of the DNA polymerase low, but synthesis of undesirable products may still occur before the start of PCR. Another solution is to delay adding the DNA polymerase until the annealing step of the first cycle. This technique is termed “hot start” since amplification can start only after the initial denaturation step above 90°C.
Although effective for improving specificity, the manual hot-start procedure is laborious and increases the risk of sample contamination and poor reproducibility. In 1994, Taq DNA polymerases with a true hot-start property were developed [1,2], where specific antibodies are bound to the polymerases to inhibit them at room temperature during the reaction setup. During the initial high-temperature denaturation step (e.g., >90°C), the bound antibodies are degraded, activating the DNA polymerases (Figure 1).
Figure 1. Antibody-based hot-start DNA polymerase and its activation in PCR to enhance specificity.
The denaturation step also separates misprimed targets and primer-dimers that may have formed during the reaction setup, thereby preventing their amplification by DNA polymerases in subsequent annealing and extension steps. In this manner, hot-start DNA polymerases reduce nonspecific amplification, increase yields, and allow convenient room temperature setup for high-throughput applications (Figures 2–4).
Figure 2. PCR results from non–hot-start vs. hot-start DNA polymerases. Note the improved yields of the desired amplicon and lack of nonspecific amplification with a hot-start DNA polymerase.
Figure 3. Suitability of hot-start DNA polymerase for room-temperature reaction setup for high-throughput applications. PCR reactions were prepared and incubated at room temperature for 0, 24, and 72 hr before loading into a thermal cycler. Highly specific amplification of a 2 kb fragment from human gDNA was observed even 72 hr after room-temperature setup, demonstrating the power of hot-start DNA polymerases for large-scale experiments.
As alternatives to antibodies, hot-start attributes can be also achieved by heat-labile chemical modifications of the enzyme’s active site, as well as by using small molecules such as aptamers to shorten the activation time. Regardless of the choice of hot-start technologies, it is crucial that the DNA polymerase’s activity be efficiently blocked under unheated conditions to ensure specificity (Figure 4).
Figure 4. Comparison of polymerase activity: (A) a true “hot-start” DNA polymerase vs. (B) a “warm-start” DNA polymerase. Polymerase activity was measured at 60°C (constant) for 60 minutes. In heat-activation tests (blue curves), polymerases were heat-treated at 94°C for 2 minutes to dissociate the antibodies from the polymerases. Without heat activation (red curves), the true hot-start DNA polymerase showed no detectable activity, whereas the warm-start enzyme displayed activation at 60°C, making it unsuitable for hot-start applications.
Since thermal cycling is a key feature of the conditions that enable the repetitive chain reaction of amplifying DNA, thermostability of the DNA polymerase to be used is an important feature. Although Taq DNA polymerase, originally derived from a thermophilic bacterial strain, can withstand relatively high temperatures, its half-life shortens significantly above 90°C. This shortfall poses a challenge when prolonged high temperatures are used to denature DNA with secondary structures and GC-rich sequences. Similarly, in amplifying long templates, Taq DNA polymerase may need to be supplied in higher amounts or replenished for prolonged incubation periods. Thus, DNA polymerases isolated from hyperthermophilic organisms have become instrumental in overcoming these challenges, due to their higher thermostability.
A well-known hyperthermostable enzyme is Pfu DNA polymerase from the archaeal hyperthermophile Pyrococcus furiosus found in hydrothermal environments. Pfu polymerase is about 20 times more stable than Taq polymerase at 95°C . Other popular hyperthermostable DNA polymerases include KOD and GBD from archaeal Thermococcus and Pyrococcus species.
Although archaeal DNA polymerases are extremely heat-stable, they may have limitations in certain scenarios. For instance, hyperthermostable Pfu DNA polymerase is slow in synthesizing DNA due to lower processivity (compared to Taq DNA polymerase). In addition, archaeal DNA polymerases are unable to amplify uracil-containing DNA templates due to the presence of a uracil-binding pocket as a DNA repair mechanism [4,5]. Uracil-containing DNA sequences are the basis of PCR carryover prevention and locus methylation analysis by bisulfite conversion.
The proofreading capability of a DNA polymerase defines fidelity, which increases the accuracy of DNA sequence replication. High-fidelity DNA polymerases are enzymes with strong proofreading activity. The ability of DNA polymerases to accurately replicate DNA sequences (i.e., attaining error-free sequences) is crucial in applications such as cloning, sequencing, and site-directed mutagenesis.
The proofreading activity of a DNA polymerase is based on its 3′ → 5′ exonuclease activity, which corrects misincorporated nucleotides. The exonuclease activity occurs at location on the DNA polymerase separate from the site of its 5′→ 3′ polymerase activity (Figure 5). When a mismatched nucleotide is incorporated at the polymerization domain, DNA synthesis stalls due to the unfavorable base-pairing kinetics. The delay allows excision of the mismatched nucleotide and its replacement with the correct nucleotide by the DNA polymerase .
Figure 5. A DNA polymerase with its 5′→3′ polymerase domain and 3′→5′ exonuclease domain (illustration based on the structure of E. coli DNA polymerase I).
The fidelity of a DNA polymerase can be measured using different methods such as colony-screening assays, Sanger sequencing, and next-generation sequencing [7-10]. Using traditional colony-screening methods, a PCR-amplified fragment of the lac gene is cloned into a plasmid. Colonies transformed with the recombinant plasmid can be assessed using blue/white colony screening. Plasmids with a mutation in the lac gene insert (presumably introduced by a replication error during PCR) result in loss of LacZ function, forming white colonies. On the other hand, the lac inserts with no PCR error result in blue colonies. Using Sanger sequencing, cloned PCR fragments can be sequenced to determine the error rate of DNA polymerases. With next-generation sequencing, PCR amplicons may be directly subjected to sequencing (Figure 6).
Figure 6. Common approaches to measuring fidelity of a DNA polymerase.
A DNA polymerase’s fidelity is frequently expressed as the inverse of the error rate (fidelity = 1/error rate), which refers to the number of misincorporated nucleotides per total number of nucleotides polymerized. Therefore, the measured fidelity of a DNA polymerase is highly dependent upon the length of PCR amplicons as well as the number of PCR cycles used in generating PCR products. To accurately compare fidelity between different polymerases, measurements must be made using the same method and cycling parameters.
Often, fidelity is expressed as relative to Taq DNA polymerase’s fidelity. The fidelity of naturally occurring proofreading DNA polymerases, such as Pfu and KOD, is around 10x Taq fidelity. However, “next-generation” high-fidelity DNA polymerases are engineered via directed evolution for exceptional fidelity, >50–100x that of Taq polymerase (Figure 7). With these enzymes, the rate of misincorporation is on the order of one in millions of nucleotides incorporated.
Figure 7. Comparison of popular high-fidelity enzymes, as determined by a next-generation sequencing method.
Processivity of an enzyme is defined as the number of nucleotides being processed in a single binding event. DNA polymerase’s processivity often reflects synthesis rate and speed, as well as affinity for its substrates. Therefore, highly processive DNA polymerases are beneficial for amplification of long templates and of sequences with secondary structures and high GC content, and in the presence of PCR inhibitors such as heparin, xylan, and humic acid, which are found in blood and plant tissues (Figure 8).
Figure 8. DNA polymerases with high processivity can efficiently amplify (A) target sequences of varying lengths from human gDNA, (B) templates with a range of GC content (without enhancers), and (C) targets from samples with common PCR inhibitors. Highly processive Invitrogen™ Platinum™ SuperFi™ DNA polymerase was used in these experiments.
Early-generation high-fidelity DNA polymerases tend to display low processivity due to their strong exonuclease activity, which slows down polymerization. Hence, amplifying long target DNA can be significantly slowed. For instance, proofreading Pfu DNA polymerase has fidelity that is 7x that of Taq DNA polymerase, but its synthesis rate is less than half that of Taq polymerase. A breakthrough in processivity was achieved when DNA polymerases were engineered with a strong DNA-binding domain of another protein without compromising polymerase activity (Figure 9) . Such engineered DNA polymerases have processivity that is enhanced 2- to 5-fold.
Figure 9. Processivity of DNA polymerases. (A) Highly processive DNA polymerases tend to have higher affinity for their substrates and incorporate more nucleotides per binding event. (B) Schematic of a DNA polymerase sequence with an engineered DNA-binding domain (Pol = polymerase domain, 3-′5′ exo = 3′→ 5′ exonuclease domain, DBD = DNA-binding domain, N = N terminus, C = C terminus).
Together, the four properties of DNA polymerases—specificity, thermostability, fidelity, and processivity—make these enzymes highly versatile, and subsequent enhancements further broaden their applications in PCR. Enzyme specificity ensures that desired PCR products are obtained in high yields while minimizing potential issues in downstream applications such as cloning and quantitation. Strong processivity and hyperthermostability overcome difficulties in amplification of secondary structures, GC-rich sequences, and long DNA. Moreover, improved processivity renders resistance to naturally occurring PCR inhibitors in DNA samples. Finally, high fidelity provides accuracy in sequence replication.
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