# Taq Polymerase's stability at high temperature

I was asked a question as to what is so special with Taq Polymerase that makes it quite stable at high temperatures though its functioning is the same as other DNA polymerases like that of mesophiles.

I just think it's the adaptation of that particular bacteria in that high-temperature environment. But I want to know if there is any strong reason for this stability.

Well, the evolutionary reason is the one you mentioned: the thermostability of Taq allowed its bacterium to colonize high-temperature environments. That, however, does not say anything about the molecular reasons for this difference.

I found this very good answer on Quora. The end of the post has several references for its claims.

Long story short, the stability of a proteins folded state depends on the difference of free energy $$Delta G$$ between the folded and unfolded states. However, $$Delta G = Delta H - T Delta S$$, which means the difference in free energy can either come from a bigger difference in enthalpy (for example, salt bridges, hydrogen bonds, hydrophobic interactions and such in the folded state) or a smaller difference in entropy (for example, a low number of "not-rigid" amino acids like glycin , or a high release of water from folding).

While enthalpy is often the reason for stability of folding, it seems like for Taq polymerase the difference actually lies in an unusually small difference in entropy between the two states.

Edit: here is one of the best references of the post. Open the link above to find more. https://www.ncbi.nlm.nih.gov/pubmed/24174290

## Purification of Thermostable DNA Polymerases

Thermostable polymerases are of course key enablers of molecular biology, due to their usefulness in polymerase chain reaction (PCR). Today I am going to show you the process by which these important enzymes are isolated. You will learn how to make your own Taq polymerase using the open source expression vector pOpenTaq, as well as understand the purity grades of the commercial polymerases offered by Gene And Cell Technologies. The principles outlined in this blog post should apply to any thermostable nucleic acid polymerase.

Taq polymerase can be expressed form pOpenTaq, an open-source taq polymerase expression plasmid. pOpenTaq should be transformed into an E. coli BL21 strain and induced with 1 mM IPTG using standard protocols. Expression overnight will maximize the yield. 100% of the protein will be expressed in soluble form at 37 o C.

Our purification strategy is going to be:

## Taq Polymerase's stability at high temperature - Biology

The component of commercial available master mix most sensitive to unfavorable conditions is a polymerase. Available commercial polymerase chain reaction (PCR) master mixes are generally recommended for storage at −20°C, otherwise they may lose their activity.

The aim of the experiment was to verify if storing mixes in adverse and extreme conditions may influence the quality of a PCR product. In the second phase of the research, it was to indicate if inactive PCR reagents that have lost their activity, may recover their enzymatic properties.

### Material and methods

Five different commercially available master mixes were incubated in unfavorable conditions. After the PCR, an electrophoresis was carried out and the obtained product was an evidence of a proper PCR reaction.

### Results and discussion

Total degradation of mixes was caused by their incubation at room temperature for 28 days and incubation at 100°C for 60 minutes. Addition of polymerases to the degraded mixes (incubation at room temperature for 28 days) resulted in a regeneration of all of five mixes. In the case of polymerases incubated at 100°C for 60 minutes, regeneration was effective only in two of the five mixes.

### Conclusions

Our research confirms that PCR master mix is characterized by high resistance to varied conditions as well as in some cases can be repaired after degradation.

## DNA Polymerase Thermostability

DNA is a dynamic molecule with a structure that is stabilized by a large number of weak interactions. The stability of the DNA double-helix depends on a variety of factors, including DNA sequence, pH, ionic strength, solvents and temperature. In particular, as the temperature is increased, the weak interactions are sequentially disrupted, first resulting in localized denaturation of the terminal and selected internal sequences, and finally resulting in complete separation of DNA strands. The degree to which this destabilization is desired or tolerated depends on the application.

For example, cloning procedures, such as end-polishing are maximized when the termini are stabilized, suggesting use of a polymerase derived from a mesophilic organism. Second strand cDNA synthesis and nick translation are other applications that traditionally use mesophilic enzymes. Such enzymes are maximally active at temperatures of 25&ndash40°C and retain significant activity at lower temperatures as well. Generally, they can be heat-inactivated and work in the same buffers used by restriction endonucleases and ligases, obviating the need for intermediate DNA purification.

A variety of other molecular biology applications, such as PCR, require high temperatures to denature the DNA prior to primer annealing or during polymerization to reduce secondary structure, thus reducing polymerase pausing. Archaeal DNA polymerases, such as Vent® (NEB #M0254) and 9°Nm&trade (NEB #M0260) are derived from hyperthermophiles and are extremely resistant to heat inactivation, even at 100°C, and display maximal polymerase activity at 75&ndash85°C. Bacterial thermophiles have yielded enzymes such as Taq DNA polymerase, which is active at similar temperatures, but is not quite as stable at 95°C, as some of its archaeal counterparts.

Vent® is a registered trademark of New England Biolabs, Inc.

## Taq Facts

When the polymerase chain reaction (PCR) was first described, the Klenow fragment derived from the Escherichia coli DNA Polymerase I was the paramount enzyme for sequence extension. Due to its lack of stability at high temperature, it needs be replenished before each cycle. Upon the discovery of thermophilic bacteria which thrive at temperatures greater than 45 °C, heat-stable polymerases which function at higher temperatures were investigated in an effort to eliminate the need to replenish enzyme following each denaturation cycle.

Taq DNA Polymerase was originally isolated from thermophilic bacterium of the Deinococcus-Thermus group located near the Lower Geyser Basin of Yellowstone National Park by Thomas D. Brock and Hudson Freeze, in 1969. This thriving bacterium was named Thermus aquaticus (T. aquaticus). Several enzymes have been isolated from T. aquaticus, the most known of which is Taq DNA polymerase (Taq). Taq can be isolated either from its original source or from its cloned gene expressed in E. coli. While many similarities in sequence and structure exist between E. coli DNA polymerase I and Taq, differences in enzyme functionality, character and dependencies make Taq the ideal polymerase for use in qPCR-based gene expression analysis, with the exception of sensitive measures of certain bacterial genes. This caveat is explained later.

Characterization of Thermus aquaticus DNA Polymerase

Functionality: Utilizing the inherent 5’ to 3’ exonuclease activity of Taq, researchers are able to simultaneously achieve PCR amplification and signal release from a target-specific fluorogenic probe. The 5’ to 3’ exonuclease activity of Taq cleaves the 5’ terminus of a hybridized oligo probe to release both mono- and oligonucleotides. The probe is hydrolyzed concomitant with strand replication so that the accumulating fluorescent signal correlates with amplification.

Size and activity: While variable weights have been reported, the approximate size of Taq is 94 kd, with the activity of a DNA polymerase localized to the C-terminus and 5’ to 3’ exonuclease activity localized to the N-terminus. To date, no 3’ to 5’ exonuclease activity has been observed as in other polymerases, where the 3’-end mismatched base is excised during a “proofreading” process. The error rate has been reported to be as low as 10 -5 for base substitution errors and 10 -6 for frameshift errors.

Temperature dependency: Thermophiles are prevalent in nature and certain prokaryotic species thrive at temperatures above 45 ° C. The temperature-dependency of Taq makes it optimum at 80 ° C, where its catalytic activity is more than ten times that typically observed at 37 ° C. It is possible that the decrease in activity above 80 ° C is actually due to the denaturation of double-stranded DNA at these temperatures.

Monovalent and divalent cation dependencies: Salt concentrations required for optimum performance are considered to be 40 mM NaCl and 60 mM KCl. Concentrations greater than 100 mM for these monovalent cations are prohibitive to catalytic activity. This is in contrast to the salt-insensitivity of the E. coli Polymerase I enzyme. In addition, Taq may depend — like other polymerases — upon the presence of divalent cations, namely MgCl2 or MnCl2, with optimum concentrations depending on the experimental design. Higher concentrations of manganese can lead to an increased error rate of nucleoside incorporation. Activity with other divalent ions may be significantly decreased or absent, as is the case with greater than 0.25 mM Ca +2 . Taq requires the presence of all four species of deoxyribonucleoside triphosphates and DNA for optimum catalytic activity.

pH dependency: The pH optimum for the enzyme is within the range of 7-8 pH units when at 80 ° C, and will vary depending on the buffer system used. In a 25 mM Tris-hydrochloride buffer for example, the alkalinity optimum is 7.8 pH units.

The great caveat: uninvited guests

On occasion, cloned Taq polymerase has been shown to have contaminating bacterial DNA that is possibly carried over from the expression vector system or other sources used during polymerase manufacture. This residual contamination may limit the use of cloned Taq in the detection of dilute bacterial DNA in certain samples. Trace contamination may be impossible to completely remove, and indeed certain estimates of contamination counts in commercially available Taq have claimed as many as 1000 genome equivalents of bacterial DNA per unit of enzyme.

Several methods for removing bacterial DNA from Taq polymerase have been tested and appear in the literature. Methods such as exposure to ultraviolet light below 320 nm (UVB or UVC) has the effect of making DNA resistant to amplification however it also affects the integrity of the Taq polymerase, reducing the efficiency of nucleoside incorporation. Ultra-filtration of the Taq polymerase, while often able to eliminate false-positives, does so with the unwanted effect of decreasing the assay sensitivity. UVA-activated 8-methoxypsoralen treatment to intercalate contaminating DNA into double-stranded DNA is difficult to optimize and inhibits the PCR reaction. In addition to the above methods, restriction endonucleases and DNAse I treatment to digest DNA in Taq preparations may introduce contaminants or become the contaminants themselves.

A recent methodology under interrogation is the serial dilution of the Taq polymerase (up to 32-fold) to effectively dilute-out the contaminating bacterial DNA while maintaining Taq activity and sensitivity. This technique has the consequence of reaction plateauing at lower cycle numbers. This effect generates a lower signal in end-point analysis, with minimal consequence to quantification based on a threshold during the exponential phase.

## Enzyme: Taq stability - (Nov/21/2011 )

I am wondering about the "strict caution" about enzyme storing at -20°C. Manufacturers often say that we have to keep enzymes (e.g Taq) upon reception at -20°C. Yet, this enzyme is used in PCR at 95°C for at least 2 h and stay active after alternative cycles of 95/55°C! This means that its stability is very high.
Also, if we look at dishwashers liquid, all enzymes are kept at room temperature and stay active!
So, my question, is my boss right to panic when I forget the Taq on bench for some minutes?

Taq and many other enzymes do degrade quite quickly at room temperature, a few minutes are probably OK, but certainly hours are not. At 95 deg taq degrades quite quickly, I think that it is only stable for a couple of minutes. The enzymes in dishwashers etc are all stabilised or are forms that are resistant to degradation. If you want to look into stable enzymes - RNase is a good place to start.

So as not to leave it at room temperature, it is good practice to keep it on ice at all times.

You say that Taq is stable for a couple of minutes, how is that in a PCR run that lasts

1h30? If it is stable only for a couple of minutes, this means that DNA will not be amplified efficiently!
During PCR run, Taq is active to amplify DNA if it is degraded, there will be no DNA amplified, or only very small quantity!

Taq from Invitrogen has this half-life times:

5 minutes at 97.5°C
40 minutes at 95°C
120 minutes at 92.5°C

I guess Taqs from other companies have similar values. Therefore higher temperatures are not that problem for a short time. But anyway this are minutes, and I'd never store it at higher temperatures or in a fridge, because the times are then days, weeks or months. Bye -20 degrees you avoid a slow, gradually degradation, but keep a more or less constant concentration of active enzyme.

Taq polymerase is quite stable at room temperature for six months providing it isn't opened! If it is repeatedly opened you stand a very good chance of culturing some nice airborne bacterial or fungal contamination in the 50% glycerol storage buffer.
(Duncan Clark, DNAmp Ltd. (Licensed PCR enzyme manufacturer) on 03.08.96 at http://www.bio.net/bionet/mm/methods/1994-. ber/019005.html)

I forgot Taq on the bench overnight.
It still worked.

It is better to keep it at -20°C, but if once you forgot it, you don't have to trash it. try with one or two samples to see if it works.
If I were your boss, I would not panic, but still I would shout on you not to forgot the enzyme on the bench.

## What are the requirements for a DNA polymerase used in PCR?

The primary requirements for a DNA polymerase used in PCR are optimal activity at temperatures around 75*C and ability to retain that activity after prolonged incubation at even higher temperatures.

#### Explanation:

PCR requires a DNA polymerase enzyme that makes new strands of DNA, using existing strands as templates.

The DNA polymerase typically used in PCR is called Taq polymerase and is isolated from the heat tolerant bacterium Thermis aquaticus. T. aquatic lives in hot springs and hydrothermal vents. It's DNA polymerase is very heat stable and is most active around 70*C. This heat stability makes Taq polymerase ideal for PCR, as high temperature is used repeatedly in PCR to denature the template DNA, or separate its strands.
Pfu DNA polymerase from Pyrococcus furiosus is also used because of its higher fidelity when copying DNA.

## 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 youve 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.

## Stabilization of Taq DNA Polymerase at High Temperature by Protein Folding Pathways From a Hyperthermophilic Archaeon, Pyrococcus furiosus

Pyrococcus furiosus, a hyperthermophilic archaeon growing optimally at 100°C, encodes three protein chaperones, a small heat shock protein (sHsp), a prefoldin (Pfd), and a chaperonin (Cpn). In this study, we report that the passive chaperones sHsp and Pfd from P. furiosus can boost the protein refolding activity of the ATP-dependent Cpn from the same hyperthermophile. The thermo-stability of Taq polymerase was significantly improved by combinations of P. furiosus chaperones, showing ongoing protein folding activity at elevated temperatures and during thermal cycling. Based on these results, we propose that the protein folding apparatus in the hyperthermophilic archaeon, P. furiosus can be utilized to enhance the durability and cost effectiveness of high temperature biocatalysts. © 2005 Wiley Periodicals, Inc.

## Taq Polymerase's stability at high temperature - Biology

MB016-HYT: High Yield Taq DNA Polymerase

* Robust processivity. Up to 10 kb human genomic and 15 kb lamda DNA fragments have been tested for amplification.
* High purity. >98% homogeneous of the Taq DNA polymerase by SDS gel electrophoresis. No contamination detected in standard PCR test reactions.
* Reproducible results. No visible activity change after storage of the high yield Taq DNA polymerase at room temperature for 3 months to amplify a single-copy gene from human genome with high efficiency.
* Proofreading with 5&prime exonuclease activity.

MB038-OS25: One-step RT-PCR Kit

The One-step RT-PCR Kit simplifies the assembly of first-strand cDNA synthesis and PCR reaction in a single tube and offers advantages of time savings, convenience, consistency, and minimal risk of contamination and pipetting errors.

MB038-TS25: Two-step RT-PCR Kit

The Two-step RT-PCR Kit has all the components required to perform first-strand cDNA synthesis followed by amplification of the cDNA product using a two-step process.

MB040-HY2: 2x HY PCR Master Mix, with blue dye

* The PCR master mix simplifies the assembly of PCR reaction and offers advantages of time savings, convenience, consistency, and minimal risk of contamination and pipetting errors.
* The high yield PCR Master Mix has increased amplification robustness, fidelity, yield, and fragment length, as well as the ability to handle difficult or &ldquodirty&rdquo templates.
* The tracking dye and precipitant have been added into the PCR PreMix so that the PCR product can be directly loaded for electrophoresis.

Features of the blend of Taq enzyme and a proofreading enzyme contained in the high yield PCR master mix:
* Robust processivity. Up to 10 kb human genomic and 15 kb lamda DNA fragments have been tested for amplification.
* High purity. >98% homogeneous of the Taq DNA polymerase by SDS gel electrophoresis. No contamination detected in standard PCR test reactions.
* Reproducible results. No visible activity change after storage of the Taq DNA polymerase at room temperature for 3 months to amplify a single-copy gene from human genome with high efficiency.
* Proofreading with 5&prime exonuclease activity.

MB041-EN-1: dNTP Mix, 10 mM each

Free of RNase and DNase and suitable for any molecular biology application that requires pure deoxynucleotides, such as PCR, DNA sequencing, cDNA synthesis and nick translation.

MB067-EQ2G / MB067-EQ2B / MB067-EQ2R: 2x PCR PreMix, with dye (green, blue, or red)

* The PCR master mix simplifies the assembly of PCR reaction and offers advantages of time savings, convenience, consistency, and minimal risk of contamination and pipetting errors.
* The tracking dye and precipitant have been added into the PreMix so that the PCR product can be directly loaded for electrophoresis.

Features of the Taq DNA polymerases contained in the PCR master mix:
* High efficiency. The extension time of the Taq DNA polymerase is shorter than 30 seconds per kb DNA. Easily amplify DNA fragments up to 5 kb.
* High purity. >98% homogeneous of the Taq DNA polymerase by SDS gel electrophoresis. No contamination detected in standard PCR test reactions.
* Reproducible results. No visible activity change after storage of the Taq DNA polymerase at room temperature for 3 months to amplify a single-copy gene from human genome with high efficiency.
* Terminal transferase activity of adding a single nucleotide (adenosine) at 3'-end of the extension product, facilitating TA cloning of PCR products.

MB042-EUT: Taq DNA Polymerase

* High efficiency. The extension time of the Taq DNA polymerase is shorter than 30 seconds per kb DNA. Easily amplify DNA fragments up to 5 kb.
* High purity. >98% homogeneous of the Taq DNA polymerase by SDS gel electrophoresis. No contamination detected in standard PCR test reactions.
* Reproducible results. No visible activity change after storage of the Taq DNA polymerase at room temperature for 3 months to amplify a single-copy gene from human genome with high efficiency.
* Terminal transferase activity of adding a single nucleotide (adenosine) at 3' end of the extension product, facilitating TA cloning of PCR products.
* Low cost: 2.5+ cents per unit. The cheapest PCR enzyme in the market.

MB066-EN-2: dNTP set, 100 mM each

Free of RNase and DNase and suitable for any molecular biology application that requires pure deoxynucleotides, such as PCR, DNA sequencing, cDNA synthesis and nick translation.

MB073-PCR: 2x Long Taq PCR MasterMix (With Dye)

For reaction set-up, all you have to do is to add templates, specific primers, and water.

MB103-11: Cell and Tissue Direct PCR Kit (rapid genomic DNA extraction coupled with PCR)

Solution based single tube protocol for rapid genomic DNA extraction
Extract genomic DNA from animal cells and tissue samples such as mouse tail and cultured cells in <10 minutes
No toxic chemicals, DNA precipitation and column purification, or proteinase digestion
Time saving, convenient, consistent, and minimal risk of contamination and pipetting errors
The tracking dye and precipitant have been added into the PreMix so that the PCR product can be directly loaded for electrophoresis.

## What is DNA Polymerase?

DNA polymerase is an enzyme which catalyzes the synthesis of DNA from nucleotides. It is the most accurate enzyme responsible for duplicating genomes and passing genetic information to offspring. During cell division, DNA polymerase duplicates all of its DNA and passes one copy to each daughter cell. In 1955, Arthur Kornberg was discovered DNA polymerase in E Coli. The function of DNA polymerase depends on several requirements template DNA, Mg +2 ions, all four types of deoxynucleotides (dATP, dTTP, dCTP and d GTP), and a short sequence of RNA (primer). Synthesis of the DNA is done in the direction of 5’to 3’ by DNA polymerase.

DNA polymerases can be grouped into seven different families: A, B, C, D, X, Y, and RT (Reverse transcriptase). Retroviruses encode for RT an unusual DNA polymerase which needs an RNA template for DNA synthesis. There are five different types of DNA polymerases found in prokaryotes for different roles in the DNA replication. DNA polymerase 3 is responsible for the polymerization of the new strand of DNA. DNA polymerase 1 is responsible for repairing and patching DNA. DNA polymerases 2, 4 and 5 are responsible for repairing and proofreading DNA. In eukaryotes, there are 15 distinct types of DNA polymerases. They include five major families.

Figure 2: DNA Polymerase

DNA polymerases are used in gene cloning, PCR, DNA sequencing, SNP detection, molecular diagnostics, etc. Taq polymerase is one kind of a DNA polymerase which can tolerate high temperatures and be available for DNA synthesis without degradation.