chemistry

Pfu DNA polymerase

Pfu DNA polymerase is a chemistry topic covered in the lgStudy science library. This page brings together a partial reference excerpt, illustrations, worked examples, real-world applications and a short study plan, so you can understand Pfu DNA polymerase rather than just read about it. In short: Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions to copy the organism's DNA during cell division (thermostable DNA polymerase). In the laboratory setting, Pfu is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step.

Key takeaways

Pfu DNA polymerase belongs to chemistry; place it in that map before memorising details.
Learn the definition first, then one example that makes the definition concrete.
Connect Pfu DNA polymerase to a quantity you can measure, compute or draw — that is where exam questions come from.
Reproduce the core statement of Pfu DNA polymerase from memory before moving on to harder problems.

Reference excerpt

Pfu DNA polymerase is an enzyme found in the hyperthermophilic archaeon Pyrococcus furiosus, where it functions to copy the organism's DNA during cell division (thermostable DNA polymerase). In the laboratory setting, Pfu is used to amplify DNA in the polymerase chain reaction (PCR), where the enzyme serves the central function of copying a new strand of DNA during each extension step. It is a family B DNA polymerase. It has an RNase H-like 3'-5' exonuclease domain, typical of B-family polymerase such as DNA polymerase II. The Pwo DNA polymerase (P61876) is 100% identical to Pfu DNA polymerase.

Features Pfu DNA polymerase has superior thermostability and proofreading properties compared with Taq DNA polymerase.

Proofreading ability Unlike Taq DNA polymerase, Pfu DNA polymerase possesses 3' to 5' exonuclease proofreading activity, meaning that as the DNA is assembled from the 5' end to 3' end, the exonuclease activity immediately removes nucleotides misincorporated at the 3' end of the growing DNA strand. Consequently, Pfu DNA polymerase-generated PCR fragments will have fewer errors than Taq-generated PCR inserts. Commercially available Pfu typically results in an error rate of 1 in 1.3 million base pairs and can yield 2.6% mutated products when amplifying 1 kb fragments using PCR. However, Pfu is slower and typically requires 1–2 minutes per cycle to amplify 1kb of DNA at 72 °C. Using Pfu DNA polymerase in PCR reactions also results in blunt-ended PCR products. Pfu DNA polymerase is hence superior to Taq DNA polymerase for techniques that require high-fidelity DNA synthesis, but can also be used in conjunction with Taq polymerase to obtain the fidelity of Pfu with the speed of Taq polymerase activity.

Uracil sensitivity This DNA polymerase, similar to other DNA polymerases from archaea is sensitive to Uracil residues in DNA and is strongly inhibited by dUTP or uracil residues in DNA. The inhibition of this class of thermostable DNA polymerases limit their use in some applications of PCR, i.e. use of dUTP for prevention of carryover contamination as well as application involving dU containing primers such as ligase free cloning methods or site directed mutagenesis using UNG.

History Scientists led by Eric Mathur at the biotech company Stratagene, based in La Jolla, California, discovered Pfu DNA polymerase which exhibits significantly higher fidelity of replication than Taq DNA polymerase in 1991. They received patents for exonuclease-deficient Pfu and the full Pfu in 1996. Other polymerases from Pyrococcus strains such as "Deep Vent" (Q51334) from strain GB-D (coding sequence 100% identical to P. kukulkanii LMOA42) have also seen use.

References

External links Stratagene's Pfu U.S. Patents Archived 2017-02-21 at the Wayback Machine Patent 5,489,523 Patent 5,545,552

Illustrations

Worked examples

Example 1 — a first encounter with Pfu DNA polymerase

Start with the simplest possible case. Write down what Pfu DNA polymerase claims or describes in one sentence, then invent the smallest concrete situation in which that sentence is true. In chemistry, the smallest case is usually a single object, a single equation or a single measurement. Check that every symbol or term in your sentence has a meaning in that case.

Example 2 — changing one variable

Take the situation from Example 1 and change exactly one quantity: double it, halve it, or set it to zero. Predict what should happen to Pfu DNA polymerase before you calculate. Comparing your prediction with the result is the fastest way to find out whether you understand the idea or only the words.

Example 3 — an exam-style question

Typical questions about Pfu DNA polymerase ask you to (a) state it precisely, (b) apply it to given data, and (c) explain a limitation. Practise writing all three answers in under five minutes; the third part is what separates a full-mark answer from an average one.

Applications of Pfu DNA polymerase

In research: Pfu DNA polymerase appears in chemistry research whenever the underlying quantities have to be modelled precisely. Papers usually cite it as a starting assumption and then explore where it breaks down.
In technology and industry: Engineering practice reuses Pfu DNA polymerase in design rules, simulations and safety margins. Knowing the idea lets you read a specification sheet and understand why the numbers look the way they do.
In the classroom: Pfu DNA polymerase is common in secondary-school and first-year university syllabi. It links to neighbouring topics DNA replication, EC 2.7.7, Polymerase chain reaction, so understanding it makes those chapters shorter.
In everyday life: Look for Pfu DNA polymerase outside the textbook — in sport, cooking, traffic, electronics or the sky above you. An example you found yourself is remembered far longer than one you were given.

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How to study Pfu DNA polymerase in 20 minutes

Read the reference excerpt below once, without taking notes.
Close the page and write down what Pfu DNA polymerase means in your own words.
Compare your version with the excerpt and mark what you missed.
Work through the three examples above with pen and paper.
Explain Pfu DNA polymerase out loud to somebody else — or to Teacher Smith in the lgStudy chat.

Frequently asked questions

What is Pfu DNA polymerase in simple terms?

Why does Pfu DNA polymerase matter?

Because it connects several chemistry ideas at once: it gives you a definition you can apply, a quantity you can calculate, and a way to check whether a result is plausible.

How should I study Pfu DNA polymerase?

Read the excerpt, restate it from memory, then work through the examples and applications listed on this page. The five-step study plan above takes about twenty minutes.

What does this page cover?

It gives you a compact reference excerpt plus original lgStudy explanations, examples, applications and study material on Pfu DNA polymerase.