Get To Know Your DNA Polymerases
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While most may think standard Taq is the backbone of PCR, many other DNA polymerase options exist. The polymerase you use significantly impacts 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!
Before we go over the different types, let’s discuss what you should look for when buying a polymerase.
What Does A Polymerase Need To Be Useful?
For starters, DNA polymerases need to be optimally active at temperatures exceeding 75°C. Next, they must not run out of steam halfway through your PCR run and should retain 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 it 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 the formation of primer dimers. Primers also tend 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). Many companies offer hot-start Taq, including New England BioLabs, Qiagen, 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.
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. 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 highly repetitive target sequence, enzyme combinations like the GC-RICH PCR System offered by Roche may also work well for you. This system uses a special solution optimized for amplifying GC-rich regions that again relies on an added proofreading polymerase.
Another option for GC-rich sequences or if you require the most faithful transcription possible is a high-fidelity hot-start polymerase such as the Q5® Hot Start High-Fidelity DNA Polymerase from NEB. These high-fidelity hot-start enzymes can be more expensive, so be sure you need them before you invest!
One of the major polymerases people turn to when they need 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.
Pfu 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 × 106 errors per base pair per duplication. The downside of Pfu is its speed which is slower than that of Taq. Combining Pfu and Taq gives you the best option as you get Taq’s speed with Pfu’s fidelity.
Overcoming dUTP Poisoning with Pfu
Pfu also suffers from dUTP poisoning, which 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. ArchaeMaxx is a dUTPase that converts poisonous dUTP to harmless dUMP (a rather wonderful name!) +iPP.
To further increase fidelity and improve elongation speed, 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 proofreading ability, so if you require fast, very high-fidelity enzymes, these are what you’ve been searching for. Examples of these include NEB’s Phusion 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 that again uses a proofreading enzyme but also special buffers. You could also 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 to amplify RNA to cDNA. This polymerase is thermostable at high temperatures (95°C for up to 20 minutes), making it very useful for reverse RNA transcription 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 is best combined with a proofreading enzyme.
Creating Blunt Ends
DNA polymerase from Thermococcus litoralis is another extremely thermostable high-fidelity option that can be used to create a product composed of about 95% blunt ends. Deep Vent® DNA Polymerase (NEB) can be used if you need thermostability of temperatures over 95°C or if the enzyme needs to be thermostable for an extended boiling period. It can also be used to create blunt ends. DeepVent is used in combination with Taq to create long sequence options. This offers an improved fidelity rate to just Taq (two-fold), as it has the proofreading 3’-5’ exonuclease abilities that Taq lacks.
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.
Do you know any more you like to use? Tell us in the comments below.
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