Archive for the ‘Taq’ Category

PCR or the Polymerase Chain Reaction

In animation, DNA, molecular biology, PCR, plasmid, Polymerase, Polymerase Chain Reaction, Replication, Taq on April 12, 2009 at 6:25 pm

When I first heard about the Polymerase Chain Reaction my first association was with the atomic bomb chain reaction. You know probably from your studies: the labile Uranium if receives a neutron it transformed to a stable Uranium isotope while several new neutrons are released. If these newly release neutrons meet novel labile Uranium atoms the reaction is amplified, more and more neutrons will be released until the system if is lost from control explodes in the form of an atomic bomb. If the reaction is under control we can produce heat and through this electricity in an electric plant, if the critical mass of the labile isotope is ignited with a neutron beam, it will explode.

You can have two excellent representations of the chain reaction below:

The chain reaction

But this is a blog about techniques in the molecular biology lab, so we will not deal with the fission chain reaction but we will see how a similar type of amplified reaction is produced with the DNA by specific enzymes in a reaction tube. The enzymes that can do a chain reaction are the Polymerases.

What is the function of polymerases? We have them in each of our cells. They do the most basic reaction that keeps life going on from the start of the very first organism ever. They are duplicating in a semi-conservative way the DNA in order to allow the transmission of the genetic information during cell division.

You can have an animation about DNA replication below.

What is the Polymerase doing?

The two DNA strands are connected by hydrogen bonds and code for the same information by the A:T and C:G base pairing.  The two strands are anti-parallel if we look to the double strand from one direction one of the strands will be in 5′-3′ direction and the other vice-versa in 3′-5′ direction. As an important rule we have to know that in nature all polymerases are doing the DNA synthesis in the 5′-3′ direction. The strand that can be replicated according to this rule is the leading strand. The other one is called lagging strand. The problem is that both strands have to replicated in by the same protein complex! But how can one single Replication complex produce the leading strand and the lagging strand in the same time ? We arrived to the problem of the the Okazaki fragments.

In the animation below you can find a good representation about how a single Replication complex can do the synthesis of the two different strands.

In order to speak about PCR we have to go out to trip to check some Geysers.

So let us check first the big Steamboat Geyser.

Yellowstone Steamboat Geyser

If we go closer to one of the hot springs we might see that the water is “living”, there are some algae, micro-organisms in this water. Let’s have a look:

The Yellowstone Hot Springs

These micro-organisms are living in really hot water. But if they are living, they should replicate, and if they replicate, they should have DNA polymerases!

These micro-organisms were isolated and one of them, called Thermo aquaticus (sometimes named Thermophilus aquaticus) became really famous. It has a polymerase that is used in vast majority of the in vitro DNA replication processes and in PCR.

I am sure you all have an idea already about PCR. We put all reagents needed for a DNA replication in a tube and reproduce the normal DNA replication process. So what do we need? We need a DNA template an oligonucleotide as a primer, the building blocks of the DNA (dATP, dTTP, dCTP, dGTP or in general dNTP -deoxi nucleotide tri phosphate), Mg and the Polymerase. If possible from Thermo aquaticus, which is called Taq.

There is one trick! This one trick was invented by Kary Mullis and he received Noble Prize for this single idea. The trick is, that we will not reproduce completely the natural reaction. We do not want to bother with leading strand and lagging strand and with all kind of Okazaki fragments and helicases and ligases.

The idea of Kary Mllis was that if you separate the double strand and design two oligos that will bind the two different strands but will look towards each other, than the product will be doubled. If you separate the strands by heating the solution to 95C you can repeat the reaction, and now you will have 4 copies. In the next run 8 copies and so on in each reaction you will have 2 on the power of the “cycle number” copies in a chain reaction fashion!!!

Let us have a really simple and good introduction to the whole procedure in the next two animations:

Below you can find a more fancy animation of the same procedure:

For this idea Mullis got the Noble Prize. His work changed completely the history of molecular biology. Let us check an interview with him about how he discovered PCR:

What is the practical use of this whole method?

Amplifying DNA by PCR became one of the most widely used method in a molecular biology lab. You can use it for transferring DNA from one plasmid to a different one, to introduce mutations and even to measure the amount of a specific gene in a sample. By combining it with a Reverse Transcription reaction we can measure copy numbers of RNA molecules.

Below we can see an example of how it is used in criminal justice!

In the next video you can see the workflow of DNA sequencing with a New Generation sequencing machine. What is remarkable here is that the designers of the instrument are skipping the cloning of the DNA fragments into plasmids and amplification of the plasmids by bacteria. They use micro reactors in the form of an emulsion PCR. One oligo is on a bead and the DNA binds the oligo. Each bead is fused with a small droplet that contains all the other reagents for the PCR. By this trick you will have a clonal amplification of the DNA fragments. One bead will contain on  type of DNA and you skipped all bacterial work. The result is that you can sequence the whole human genome in a couple of weeks for less the 100k USD. Or you can sequence a bacteria in a day…

Emulsion PCR in the FLX sequencer workflow

At the end of this lesson, lets have some fun and see the celebration of the PCR!

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