Pfu DNA Polymerase: Highly Accurate and Reliable Enzyme

28 June 2024

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Pfu DNA Polymerase comes from the super hot archaeon Pyrococcus furiosus. Family B, it’s really good at making DNA without mistakes.¹ This enzyme is perfect for projects needing exact DNA copies. Projects like cloning, changing specific DNA spots, and studying how genes work. Its special traits make it a top pick for a lot of DNA work.

Key Takeaways:
– Pfu DNA Polymerase is a highly thermostable and accurate DNA polymerase isolated from the hyperthermophilic archaeon Pyrococcus furiosus.
– It exhibits superior fidelity and proofreading ability compared to other polymerases like Taq.
– Pfu DNA Polymerase is a valuable tool for high-fidelity PCR applications that require precise DNA synthesis.
– The enzyme’s unique features make it a reliable and versatile choice for a wide range of molecular biology applications.
– Pfu DNA Polymerase’s exceptional thermostability and proofreading ability make it well-suited for applications such as cloning, site-directed mutagenesis, and expression studies.

Introduction to Pfu DNA Polymerase

What is Pfu DNA Polymerase?

Pfu DNA Polymerase is a special enzyme from the bacteria Pyrococcus furiosus². It belongs to the DNA polymerase B-family. This enzyme is very stable at high temperatures and can fix mistakes it makes, allowing accurate copying of DNA³.

Origins and Discovery

In 1991, Eric Mathur and his team found Pfu Polymerase at a company in La Jolla, California². They got it from a bacteria that loves extremely hot places. This special enzyme was much better at accurately copying DNA than the usual one, Taq DNA Polymerase².

Key Features of Pfu DNA Polymerase

Pfu Polymerase stands out for its great features. It’s a key tool in labs for working with DNA. It has top-notch traits that help in making many copies of DNA.

High Thermostability

This enzyme can work at super hot temperatures, even up to 100°C.⁴ Such ability is perfect for copying DNAs, even the hard ones. DNAs that are super detailed can be copied well because of this.

Superior Fidelity and Proofreading Ability

Pfu DNA Polymerase can fix mistakes in DNA copying, thanks to a special activity it has.⁴ It makes less mistakes than other DNA tools, almost no errors at all.⁴ Because of this, it’s ideal for important DNA work that needs to be very accurate.

Blunt-End PCR Products

Pfu Polymerase makes DNA pieces that are cut smooth at the ends. It’s great for a certain type of DNA work. With this feature, work on DNA gets simpler, saving time.

Applications of Pfu DNA Polymerase

Pfu DNA Polymerase is the top pick for high-fidelity PCR work. It offers precise DNA copying with a very low error rate. This makes it perfect for tasks like cloning, sequencing, and site-directed mutagenesis. In these, keeping the copied DNA accurate is key.⁶

Cloning and Expression Studies

The blunt-ended PCR products from Pfu DNA Polymerase fit well for cloning without working too much. They easily join with vectors, saving time and effort. Pfu DNA Polymerase is crucial for cloning and expression studies. Here, getting the DNA sequence right matters a lot.⁶

Site-Directed Mutagenesis

For tweaking specific DNA parts in site-directed mutagenesis, Pfu Polymerase shines. Its high accuracy level and proofreading catch slight errors. This leads to straight-on changes in the target DNA during amplification.⁶

Comparison with Taq DNA Polymerase

In the world of DNA amplification, Pfu Polymerase does better than Taq. It makes fewer mistakes and is more accurate. Pfu’s error rate is about 1.3 x 10^(-6) mutations per base pair per round of copying and Taq’s is 8.0 x 10^(-6).⁷ Because of this, Pfu is chosen when high accuracy is needed.

Fidelity and Error Rates

Taq used to make a mistake every 9,000 bases,⁸ but Pfu’s mistakes are rare, happening about once in 1.3 million base pairs.⁸ Experiments found that adding 2-3 mM MgSO4, 100-300 microM of each dNTP, and keeping the pH at 8.5-9.1 boosts Pfu’s accuracy even more.⁷ The exo-Pfu version, though, had errors that were 40 times more than the original Pfu under perfect conditions.⁷

Thermostability

Pfu Polymerase can handle heat better than Taq. This means it can be used for DNA that needs higher copying temperatures or longer times. It’s great for copying templates that are rich in GC.

PCR Product Specificity

The types of DNA made by Pfu and Taq are different. Pfu makes DNA that’s blunt at the ends, but Taq’s has a special structure at one end. For cloning, Pfu’s type is easier to work with because it doesn’t require extra steps.

Feature	Pfu DNA Polymerase	Taq DNA Polymerase
Error Rate	1.3 x 10^(-6) mutations/base pair/duplication⁷	8.0 x 10^(-6) mutations/base pair/duplication⁷
Thermostability	Highly thermostable, withstands higher temperatures⁸	Thermostable
PCR Product	Blunt-ended	3′ A-overhangs
Extension Speed	20 kb/min, 1-2 min/kb⁸	1-2 kb/min⁸
Fidelity	20x⁸	18x⁸

Optimizing PCR with Pfu DNA Polymerase

When working with Pfu in PCR, you need to think about the ingredients and their amounts. This means looking at how much template DNA, dNTPs, primers, and MgSO4 you use.⁹ All these things affect how well and accurately PCR will copy your DNA. By adjusting these elements just right, you can make sure your DNA amplification is successful with Pfu DNA Polymerase.

Reaction Components and Concentrations

To get the best results, the steps in PCR using Pfu must be set just right. This includes the time to heat the DNA at the start, how long to keep it hot, how long to cool it, and the time to add new DNA pieces.⁹ Pfu works well with longer times for adding new DNA parts, especially for copying long sections of DNA.

Cycling Parameters

It’s very important to pick the right primers when you’re using Pfu Polymerase.¹⁰This means looking at how long the primers are, what they’re made of, and making sure they don’t stick to themselves or to other places they shouldn’t. Good primer design helps Pfu Polymerase copy your DNA precisely and efficiently.

Primer Design Considerations

Determining the best amount of Pfu Polymerase for your PCR is key. Normally, you’d use between 2.5 to 5.0 U of the enzyme for every 100 microliters, if your DNA piece is under 2 kb.⁹ It’s also crucial to keep the Mg2+ above 1.5 mM and the pH high. For the best results, aim for dNTP concentrations between 100 and 250 μM.

The error rate for Pfu is very low, about 1.3 in a million. This leads to only 2.6% of the copied DNA being wrong.⁹ In comparison, Taq DNA Polymerase makes more mistakes, with an error rate of 8 in a million and 16% of the DNA being incorrect. This is why Pfu is often preferred when making sure the copied DNA is accurate.

Commercial Availability and Formats

Pfu Polymerase is available from companies like Thermo Fisher Scientific and New England Biolabs. They offer it in different sizes and types. This variety meets the needs of scientists and labs. You can get the original Pfu enzyme or new kinds designed for specific uses.

It comes in 100u and 500u sizes¹¹. The 100u size costs €135,00¹¹. The 10X Reaction Buffer includes 20mM MgSO4 and is 1.2ml¹¹. Its Unit Definition is how much can make 10nmol of dNTPs in 30 minutes at 75°C¹¹.

Scientists use Pfu in many ways. For example, they make human naïve antibody gene libraries and clone genes for viral antibodies¹¹. With a Performance Guarantee, you can expect excellent results from Promega PCR products¹¹. Key studies in virology, molecular biology, and genomics have used Pfu DNA Polymerase¹¹.

Protocols and Guidelines

When using Pfu DNA Polymerase for PCR, follow strict rules to prevent contamination.⁴ You should work on DNA samples, set up PCR, and analyze them in different spots. Use special tools and chemicals. Also, use UV-irradiated workstations and special pipette tips to avoid contamination.

Preventing PCR Contamination

To keep your work clean with Pfu DNA Polymerase, follow key steps. Keep the areas for before and after PCR separate. Use only certain lab tools and precautions like UV light and special pipette tips. This lessens the chance of mixing up samples.⁴

Reaction Setup

The way you set up your PCR with Pfu DNA Polymerase is critical.¹² Add the enzyme last. Use the right amounts of DNA, primers, dNTPs, and MgSO4. Make sure your machine’s settings match what Pfu DNA Polymerase needs.¹²

Selecting and measuring the right ingredients is vital for the PCR to work well with Pfu DNA Polymerase.¹² You need to find the best levels for DNA, dNTPs, and primers, plus the right amount of Mg. This is key for your PCR to be successful.¹²

Also, the times and temperatures for each PCR step need to be perfect.¹² This means how long to heat the DNA, cool it, and copy it. Getting these settings right makes Pfu DNA Polymerase work its best.¹²

Choosing the right primers is also important with Pfu Polymerase. Pay attention to the size and makeup of the primers. Avoiding certain pairing and location mistakes is crucial for the primers to attach well and make DNA copies right.¹²

Variations and Modifications

Scientists mix¹³ Pfu Polymerase with Taq DNA Polymerase to get the best of both. Blend formulations are made to speed up DNA amplification. This way, they can work faster without losing accuracy.

Engineered Variants

Aside from the usual Pfu Polymerase, there are new versions. These have better heat resistance, more processivity, or special uses. Modified Pfu DNA Polymerases offer more benefits for different studies.

Historical Significance and Milestones

In 1991, Eric Mathur’s team at Stratagene found Pfu Polymerase. This was a big step in molecular biology.¹⁴ The DNA polymerase, from the archaeon Pyrococcus furiosus, is both accurate and stable. It helped scientists work better in life science research.

In 1991, Pfu Polymerase changed the game. It came with a proofreading function to fix errors. This added accuracy and made DNA research more reliable.¹⁴ The polymerase was stable even at high temperatures. It became essential for many DNA amplification tasks.

Then, in 2003, Phusion DNA Polymerase and later Phusion Plus DNA Polymerase came into play. Thermo Scientific developed them, showing how PCR tools kept getting better.¹⁴ These advancements highlighted Pfu DNA Polymerase’s importance in molecular biology’s growth.

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