Tips on Choosing the Right DNA Polymerase

When cells divide, they need DNA polymerase to catalyze the synthesis of the new DNA strand from deoxyribonucleotides to create a copy of the original template. DNA polymerases are also used for DNA amplification using polymerase chain reaction (PCR) for diverse applications like cloning, sequencing, and more. There are many types of DNA polymerases, and each one has properties suitable for certain applications. Some of these key properties include specificity, thermal stability, fidelity, processivity, and elongation rate, which can be altered to varying extents depending on the application. Using the right DNA polymerase is extremely crucial, as it can impact downstream outcomes such as specificity, efficiency, multiplexing, automation, and simplicity of the PCR assay or application.

Matching polymerase properties to existing needs

When selecting a DNA polymerase, the first thing one has to think about is the purpose, says Pedro Quintas, Molecular Biologist at PCR Biosystems. “Is it to detect a sequence of DNA or is it to amplify DNA for a downstream application?” For DNA amplification, a proofreading polymerase and one with high processivity is desirable, depending on the size of the target. For DNA detection, proofreading is not necessary. “For isothermal amplification you want a polymerase capable of strand displacement but without the 5′ to 3′ exonuclease activity [proofreading activity],” says Quintas. “You do want 5′ to 3′ exonuclease activity if you are using hydrolysis probes for detection.”

You do need to pick a DNA polymerase with proofreading activity if you plan to clone or express your amplified product, says Natascha Buter, Technical Services Scientist, Promega. “It is also important to remember that due to its 3’ to 5’ exonuclease activity, a proofreading DNA polymerase will not leave an A overhang at the end of your PCR product that you might need for TA cloning.”

The DNA polymerase commonly used in PCR is Taq polymerase, isolated from the bacterium Thermus aquaticus. According to Yashaswini Jalikop, Product Manager, PCR Molecular Reagents & Tools, at MilliporeSigma, Taq DNA polymerase is good if the purpose of amplification is to confirm the presence of a specific sequence, such as the presence of a desired insert in a plasmid, a standard, or colony PCR. However, if the purpose of amplification is to use the PCR product for subsequent experiments, such as cloning, mutation analysis, protein expression, or next-generation sequencing where accuracy is crucial, a high-fidelity (Hi-Fi) polymerase is preferred. They exhibit strong 3' to 5' exonuclease activity, an activity that Taq DNA polymerase lacks, and are ideal for GC-rich genes and for the amplification of long templates and templates containing uracil. “Although Taq DNA polymerase is highly thermostable, its half-life reduces significantly at temperatures above 900C. This can be a challenge when working with long DNA templates or difficult templates with high GC-rich regions or secondary structures. In order to overcome this problem, it is important to use polymerases with increased thermostability,” says Jalikop.

Factors to Consider before Choosing a DNA Polymerase

• Thermostability—how long can the DNA polymerase tolerate high temperatures required for specific PCR cycling conditions and still function at its best
• Processivity—how many nucleotides can the DNA polymerase incorporate during synthesis of the new strand before disassociating
• Speed or extension rate—how fast does the DNA polymerase synthesize the new strand, often tied to processivity
• Specificity—how many non-specific or unwanted (background) targets are amplified in addition to your desired target, or instead of your desired target
• Fidelity—does the DNA polymerase have 3’ to 5’ exonuclease or proofreading activity that allows it to correct mistakes by removing and replacing mismatched nucleotides
• Robustness—how well does the enzyme work with a particular sample type
• Efficiency—how well does the polymerase overcome PCR inhibitors when working with crude samples, or how efficiently does it amplify GC- and AT-rich DNA

Sources: Natascha Buter, Promega, and Elizabeth Carpenter, Roche Diagnostics

Setting up a PCR at room temperature can lead to non-specific amplification due to primers annealing to non-complementary DNA sequences or forming primer dimers ahead of PCR cycling. The DNA polymerase then amplifies these non-specific products, leading to erroneous results. “Non-specific amplification is especially problematic in situations where there is complex or dilute template DNA, large number of thermal cycles (>35), or multiplex primer pairs,” says Jalikop. To address the problem of non-specific amplification, methods like Hot Start (HS) PCR have been developed where the polymerase activity is inhibited at ambient temperatures through various mechanisms such as reversible chemical modification or inhibition using antibody or aptamers. When the appropriate temperature is reached during PCR cycling, the enzyme gets modified or dissociates from the inhibitor and begins polymerization. “Hot Start DNA polymerases are preferred in multiplex PCR and in PCR assays with a low amount of starting material, as it reduces non-specific amplification, generation of primer-dimers, and increases the yield of the desired fragment.”

Important Functions of DNA Polymerases

3′–>5′ Exonuclease activity: The polymerase is capable of cleaving nucleotides that were just introduced and correct errors (proofreading).

5′–>3′ Exonuclease activity: The polymerase is capable of cleaving nucleotides in the direction of polymerization, which allows it to perform DNA repair. This activity is also used to remove attached probes when the complementary strand is synthesized.

Strand displacement: The polymerase is capable of displacing downstream DNA encountered during synthesis. If the polymerase also has 5′–>3′ exonuclease activity, the displaced DNA is destroyed. If not, it is kept intact.

dU tolerance: The polymerase can use templates containing uracil or is capable of using dUTP during polymerization. Addition of uracil is a common technique to prevent cross-contamination.

Resulting ends: The polymerase creates blunt ends or adenine overhangs. This is relevant for downstream applications such as cloning.

Source: Pedro Quintas, Molecular Biologist at PCR Biosystems

“Determining what end result to expect is a good starting point for evaluating DNA polymerases,” says Elizabeth Carpenter, Senior Support Scientist, Roche Diagnostics. “This helps narrow down enzymes by the level of fidelity needed. Then you can look into the extent of multiplexing required for the assay and size of the amplicon.” While selecting the appropriate enzyme for an assay, it’s also important to understand how the buffer content affects the results. “For example, buffer composition can impact the appropriate primer annealing temperature,” says Carpenter. It is also important to consider the DNA polymerase format—whether to choose a stand-alone enzyme, a lyophilization-compatible formulation, or a master mix for ease-of-use and convenience.

What to Do Before Choosing the DNA Polymerase for Your Application

Before identifying the DNA polymerase, you should ask:

• Do I need a DNA polymerase that has proofreading activity because I am cloning, using my product for expression, or performing mutational analysis?
• Am I trying to amplify a long target?
• Am I trying to identify a genotype with high accuracy?
• What costs do I need to consider?

After identifying the DNA polymerase, you should:

• Ask colleagues if they have used any of the choices being considered
• Ask a technical representative of the company who offers the DNA polymerase of your choice for advice
• Look at the external literature
• If you are trying to replace a DNA polymerase that you have successfully used in the past, compare both enzymes with a target and primer set that you know works
• Re-optimize cycling conditions and reaction set-up, if needed

Source: Natascha Buter, Technical Services Scientist, Promega

Improving polymerase properties to meet emerging demands

As genomic and diagnostic applications demand faster and more accurate results, most of the improvements in DNA polymerases now focus on fidelity, processivity, and speed. “As PCR is becoming the preferred choice assay in clinical diagnostics, there is a need for ultra-fast polymerases that are resistant to inhibitors typically found in crude biological samples, such as blood samples, body fluids, or tissue,” says Jalikop. “There is huge potential in the diagnostic and point-of-care markets for DNA polymerases suitable for isothermal amplification methods, which are not restricted by the limitations of thermal cycling. Emerging methods such as loop-mediated isothermal amplification (LAMP) and rolling circle amplification require unique DNA polymerases, such as Bst and Phi29, that have the ability to separate DNA strands of a duplex by strand-displacement method rather than by heat denaturation.”

Carpenter agrees that enhancing the ability of a polymerase to tolerate common PCR inhibitors and better multiplexing capabilities, especially for applications using difficult sample types, is going to be crucial. It’s also important to have the enzymes available as Ready Mixes, where the enzymes and buffer components are premixed for an easy ready-to-go assay. This increases convenience of use and enables the end-user to have more control over assay setup. “A polymerase that can utilize both DNA and RNA as templates, allowing for amplification of RNA without requiring reverse transcriptase, is also in demand for both clinical and research applications,” says Jalikop. Lyophilization-friendly formats of polymerase that can be freeze-dried and are stable at ambient temperatures are also gaining popularity.

Long and accurate PCR is achieved by combining a highly processive thermostable polymerase with a second thermostable polymerase that exhibits a 3′ to 5′ exonuclease activity. This blending dramatically increases the length of amplification by using the proofreading polymerase to repair terminal misincorporations that can cause a terminal arrest of elongation. This repair allows the polymerase to resume elongating the growing DNA strand and is ideal for applications such as genome mapping and sequencing and rapid identification and cloning of complete genes from genomic DNA. “However, fidelity is compromised as a result of this blend of two enzymes, and we have developed a single enzyme that enables high fidelity amplification of long or GC-rich templates (complex templates up to 17.5kb and over), which is particularly useful in sequencing workflows,” says Matteo Beretta, Molecular Biologist, PCR Biosystems.

“Another factor that influences PCR outcome is the elongation rate or the speed at which the nucleotides are added. Many fast DNA polymerases are now commercially available, usually supplied with an optimized buffer or as a master mix,” says Jalikop. Polymerases have also been designed for rapid extraction of genomic DNA from a wide variety of cells and tissues such as mouse tails, buccal swabs, saliva, and hair shafts, followed by direct DNA amplification from the extract. “There are always new ‘variants’ obtained by engineering polymerases to increase their speed, processivity, fidelity, specificity, range of templates, and range of applications, and I do not expect this to stop,” says Beretta.

Common Misconceptions When Using DNA Polymerases

There are some common mistakes made when using DNA polymerases and many of them are associated with the notion that reagents can be used inter-changeably. With many engineered variants of polymerases being created to improve fidelity, specificity, and other factors, it is very likely that even the Taq polymerase from different manufacturers may not be exactly identical. Therefore, it is extremely important to use the buffer system provided with a particular polymerase. Mix and match may not always work!

Here are a few examples that highlight some common causes of error:

• Speed of reaction: Family B members (e.g. Pfu) tend to be slower than family A members (e.g, Taq) as they possess a proofreading activity
• Overhangs: enzyme lacking 3’–5’ exonuclease activity (e.g. Taq) would add an extra adenine at the 3’ end, particularly useful for faster cloning strategies not involving the use of restriction enzymes, while a proofreading enzyme would not
• Precision: Due to the proofreading activity, enzymes belonging to family B have ~ 100x lower probability of incorporating mistakes while amplifying DNA. However, the rate of mistakes made is not excessively high in family A enzymes, and hence, they can be used for short and fast amplifications
• Template specificity: an enzyme belonging to family RT cannot be used to amplify DNA, and enzymes belonging to families A and B cannot be used to amplify RNA (unless they are re-engineered).
• Optimal temperature of reaction: Polymerases come from different sources and have a broad range of reaction temperatures. Enzymes used for classic PCR reactions (Taq, Pfu) quite often are from archea bacteria (as they need to stand high temperatures) and will work similarly, but if a Klenow fragment or Bst polymerase is used, they will not work in normal PCR setups, as their optimal temperature is 37°C and 65°C, respectively.

Source: Matteo Beretta, Molecular Biologist at PCR Biosystems

For more information on DNA polymerases, check out this related article: Getting to Know the DNA Polymerase Family