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Learning outcomes
At the end of the practice, students will acquire the following learning outcomes:
Knowledge:
1. They will be familiar with the principles of PCR.
2. They will be familiar with the ingredients and the steps of the PCR.
3. They will know the applications of PCR.
Skill:
4. They will be able to prepare a PCR reaction.
Attitude:
5. They will inquire about the background of the PCR reaction.
Responsibility and autonomy:
6. They will be able to make and use master mixes on their own.
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Testing the polymerase activity of the extracted Pfu
During the previous practices, we transformed the pET-Pfu plasmid into Rosetta cells, then we applied IPTG and lactose to induce the protein production in these cells. As a next step, we disrupted the cells by sonication and heat treated the cell extract to precipitate the non-thermostable proteins. We pelleted the non-non-thermostable host proteins to separate them from the induced heterologous protein by centrifugation.
We determined whether the purification of the Pfu DNA polymerase was successful by analysing our samples on polyacrylamide gel. The aim of this practice is to test the activity of the purified Pfu DNA polymerase.
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Theoretical background
For testing the biological activity of the Pfu DNA polymerase we are going to use the polymerase chain reaction (PCR). PCR makes the quick amplification of relatively short DNA fragments in vitro (in test tubes) possible. The invention of this technique in the early eighties by Kary B Mullis had a huge impact on life sciences, and nowadays PCR is used in many-many research- and biotechnology-related applications.
PCR is similar to the replication process in living cells, in which DNA template is used to generate new copies of the DNA by a polymerase enzyme. During PCR reaction, the double stranded DNA is getting single stranded (denaturation), then primers attach to it (annealing) and a DNA polymerase is used to extend the attached primers while reforming the double stranded DNA molecules again (polymerisation/extension). However, instead of using helicases (as in nature), during PCR heat is responsible for the denaturation of the double stranded DNA and instead of RNA primers (synthesized by the primases during replication), during PCR single stranded DNA oligomers are added to the reaction to make the start of DNA synthesis possible. For PCR, specific thermostable DNA polymerase is used, and the steps of this reaction are repeated in a cyclic program.
The required materials for a PCR
Six basic components are needed for a PCR.
First of all, we need a template, which can be a mixture of DNA molecules containing the DNA region, which needs to be amplified.
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Generally, by a PCR reaction, we can copy a relatively short fragment of DNA. (Fortunately, we almost never need to or want to amplify very long template molecules. For example, handling a whole chromosome would be rather challenging.)
We also need primers because DNA polymerases cannot start DNA synthesis, but they can extend existing DNA molecules by adding new nucleotides to their 3’ ends. Primers used in PCR are short single stranded DNA oligonucleotides (15-25 nucleotides) that are complementary with regions of the template molecules. Therefore, primers could hybridize to the template molecule and provide a docking surface for DNA polymerase. During PCR, two primers are used. These determine the borders, between which the DNA synthesis is performed.
One is called forward and the other is called reverse primer. They are complementary to different strands of the template molecule. When the primers hybridise to the template strands, their 3’ ends face towards each other, therefore the PCR product will be the region, which is located between the attachment surfaces of the primers on the template strand (see more at the end of this chapter).
For PCR reactions we also need the “building blocks” of the DNA molecule, the nucleotides: these are incorporated by the polymerise enzyme from nucleoside-triphosphates - dATP (d: deoxiribo; A:
adenosine; T:tri; P: phosphate), dGTP (G: guanosine), dCTP (C:
cytidine), and dTTP (T: thymidine). These molecules serve not only as building blocks but also provide the energy required for DNA synthesis:
the cleavage of pyrophosphate group (PPi) from them provides the energy for phosphodiester bond formations. These precursor molecules are added to the reaction as a mixture labelled as dNTP (N: nucleoside)
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mix. During synthesis, the 3’ OH of the growing chain of DNA attacks the α-phosphate on the next dNTP to be incorporated, resulting in a phosphodiester linkage and the release of a pyrophosphate (PPi).
DNA polymerase is the enzyme that performs the synthesis reactions.
Since during PCR, denaturation (the separation of the DNA strands) is performed at a high temperature, thermostable enzymes should be used since it can preserve its activity even under these conditions. Such enzymes could be isolated from extreme thermophile organisms, mainly from archaea. The most well-known polymerase used for PCR was isolated from the archeon Thermus aquaticus, therefore it was named Taq DNA polymerase. Taq polymerase is a low fidelity polymerase, since it has no proofreading activity (3’-5’ exonuclease activity). The Pfu polymerase, which was isolated from Pyrococcus furiosus, has higher fidelity than Taq polymerase, because it has a 3’-5’ exonuclease activity. By this activity, nucleotide incorporation is double checked by the enzyme and in case of a wrong nucleotide incorporation into the growing DNA strand, the enzyme can immediately remove it. This increases the fidelity of DNA synthesis. Consequently, Taq-related DNA synthesis results in more errors in the DNA, than Pfu-related synthesis does. In addition to these two enzymes, many other polymerases are used for PCR and among them, there are some engineered versions produced by recombinant DNA technology (for example, DreamTaq and Phusion have been made from Taq and Pfu, respectively).
(The polymerase gene with which we work during this practical course was cloned by us from DNA isolated from Pyrococcus furiosus cells obtained from hot springs of the Yellowstone National Park US.)