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Get To Know Your DNA Polymerases

Get To Know Your DNA Polymerases

While most may think standard Taq is the backbone of PCR, there are many other DNA polymerase options out there. The polymerase you use has a significant impact on the efficacy of your PCR, specifically on the product yield, the purity of the product and the faithfulness with which the starting product is transcribed. Sometimes, these matter less, and quick and dirty PCR is all you need, but if they do, you have several other options!

Let’s go over the different types but first, when buying a polymerase, what do you look for?

What Does A Polymerase Need To Be Useful?

For starters, it needs to be optimally active at temperatures exceeding 75°C. Next, it must not run out of steam halfway through your PCR run, it should retain its activity after prolonged incubation. It should also be thermostable at higher temperatures of around 95°C, otherwise when you are denaturing your DNA your enzyme will become useless! Lastly, look at its ability to proofread and check it is the best option for your money, for the type of PCR you’re doing and the quality of the product you require.

Once You Go Taq You Never Come Back

Why is Taq so popular you may ask? Well for starters it got there first! It also works well for simple, conventional PCR.

Taq polymerase was named after the thermophilic bacterium Thermus aquaticus from which it was isolated in 1965 by Thomas D. Brock. Its ability to withstand temperatures of up to 97.5°C with an optimum activity range of 75-80°C made it ideal for PCR and thus is has become the standard polymerase used in PCR reactions.

The Downsides to Taq Polymerase

Unfortunately, the activity of Taq peaks at 72°C but is active at lower temperatures, which can encourage formation of primer dimers. Primers also have a tendency to bind non-specifically at low temperatures, which can result in non-specific products in your PCR reaction, and standard Taq only encourages this sort of behavior! For this reason, standard Taq is best used with trusted PCR protocols that don’t call for reams of optimization.

Standard Taq is also not particularly accurate, with reported error rates of between 1.1 x 10-4 errors per base pair per duplication and 8.9 x 10-5 errors per base pair per duplication, so if you’re looking for a high fidelity product, look elsewhere.

If You Can’t Face Letting Your Taq Go

Taq advocates (Taqvocates?!) will be pleased to know that a solution is available to the over-eagerness of Taq: HotStart Taq a.k.a. FastStart Taq. Hot Start Taq DNA Polymerase is a mixture of Taq DNA Polymerase and an aptamer-based inhibitor. This inhibitor is bound reversibly to the enzyme, inhibiting its polymerase activity at temperatures below 45°C. Once this temperature has been reached, the inhibitor releases the enzyme. This means that HotStart Taq has no activity at lower temperatures. A separate activation step to release the enzyme is not required as this reaction is incorporated into the 30-40 cycles of the PCR at the beginning (95°C, 2-4 minutes). There are many companies that offer hot-start Taq including New England BioLabs, Qiagen, Life Technologies and Promega. HotStart Taq option is extremely popular and can be used for most types of PCR, but if you have longer sequences, GC-rich sequences or require extremely high fidelity, read on.

For completeness, I’ll also mention another option: hot-start PCR.

Enzyme Combinations

While standard Taq isn’t a good proofreader and will leave typos in your product, many companies are now selling Taq mixed with a thermostable, proofreading polymerase, such as Toyobo’s and Life Science’s. These mixtures are a great option for targets that are on the longer side (up to 5 kb).

If you’ve got a GC-rich target sequence or a target sequence that is highly repetitive, enzyme combinations may also work well for you, such as this one by Roche. This system also uses a special solution optimized for amplification of GC rich regions that again relies on an added proofreading polymerase.

Another option for GC-rich sequences and also for cloning multiple targets in a single reaction (multiplex PCR) or if you require the most faithful transcription possible, is a high-fidelity hot-start polymerase such as this one by New England BioLabs. They can be more expensive so be sure you need it before you invest!

Pfu Polymerases

One of the major polymerases people turn to when they are in need of high fidelity is Pfu (Pyrococcus furiosus) DNA Polymerase. These polymerases are isolated from Pyrococcus furiosus, an extremophilic species of Archaea that thrive under extremely high temperatures. This polymerase is ideal for individually cloning products for sequencing, mutagenesis or expression experiments. Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3′ to 5′ exonuclease proofreading activity, meaning that it works its way along the DNA from the 5′ end to the 3′ end and corrects nucleotide-misincorporation errors. This means that Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts, with a published error rate of around 1.3 × 10?6 errors per base pair per duplication. The downside of Pfu is its speed which is slower that that of Taq. Combining Pfu and Taq gives you the best option as you get the speed of Taq with the fidelity of Pfu.

Overcoming dUTP Poisoning with Pfu

Pfu also suffers from dUTP poisoning and is remedied with a variant called Pfu Turbo (Pfu and ArchaeMaxx factor combination). dUTP poisoning is caused by the accumulation of dUTP by dCTP deamination and results in decreased proofreading by your enzyme. ArchaeMaxx is a dUTPase that converting poisonous dUTP to harmless dUMP (a rather wonderful name!) +iPP

Engineered Enzymes

In order to further increase fidelity and also to improve on the speed of elongation, newer forms of polymerases have been created. Many of these designer enzymes are based upon or are modified forms of Pfu DNA polymerase. The modifications have allowed the speed of the enzyme to be increased as well as the proof-reading ability, so if you are in need of fast, very high fidelity enzymes these are what you;ve been searching for. Examples of these include NEB’s Phusion polymerase, Gene-On’s One-Fusion DNA Polymerase and Agilent’s PfuUltra II Fusion HS DNA Polymerase.

Dealing with Long PCR products

If you have a ridiculously long sequence of up to 25 kb don’t fret, there are long range concoctions available, such as this one by Roche that again uses a proofreading enzyme but also special buffers or you can try using a Stoffel fragment, made from a truncated Taq gene that codes for a protein lacking 5′-3′ exonuclease activity allowing it to amplify longer targets than the native enzyme.

RNA Amplification by PCR

Tth polymerase (Thermus thermophilus HB-8) has reverse transcriptase activity when mixed with manganese ions and can thus be used for the amplification of RNA to cDNA. This polymerase is thermostable at high temperatures (95°C for up to 20 minutes), making it very useful for RT of RNA where secondary structure can be a problem. However, it also lacks 3′?5′ exonuclease activity meaning it doesn’t have the capacity to proofread in this direction and so is best combined with a proofreading enzyme. There are lots of companies that offer this including Promega, Roche and affymetrix.

Creating Blunt Ends

Vent® (Thermococcus litoralis) is another polymerase that is extremely thermostable high-fidelity option that can be used to create a product composed of about 95% blunt ends. DeepVent™ can be used if you need thermostability of temperatures over 95°C or if the enzyme needs to be thermostable for extended period of boiling and it can also be used to create blunt ends. DeepVent is used in combination with Taq to create long sequence options and is made by New England BioLabs.This offers an improved fidelity rate to just Taq (two-fold), as it has the proofreading 3’-5’ exonuclease abilities that Taq lacks. Life Sciences have something similar to Vent.

Short Target Sequences

Pwo (Pyrococcus woesei) is a lesser known option and works well for sequences under 3 kb. Pwo breaks when reaching erroneous uracils (U) in DNA from the chain extension and, through this read ahead function, fewer defective DNA clones are synthesized. Pwo is available from Roche and Genaxis.

Do you know any more you like to use? Tell us in the comments below.

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Image Credit: Steve Jurvetson

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