School project #1: PCR

Hi there!

My first school project post will be about PCR. I will tell about the technique and where they use it. But first things first, what does it mean?

PCR: a technique that copies a segment of DNA until you have enough DNA copies to test its presence. Most of the time they test it using the electrophoresis technique.

Technique

The PCR-reaction takes place in a device that can heat up and cool down samples accurately, and repeat that cycle for as long as you’d like. You just have to put the sample in the device and select the temperatures you want the cycle to have.

The mixture contains:

  • DNA sample
  • DNA-polymerase
  • Two specific primers (this is different for every test)
  • dNTP’s (dATP, dCTP, dGTP and dTTP)
  • Buffer
  • Magnesium chloride
  • Enzyme (example: Taq-enzyme)

The reaction has three phases:

  1. Denaturation: the DNA in the test tube is heated up to its optimal temperature. Because of the heat, the double helix shape of the DNA falls apart, so you have two flat strings of base pairs.
  2. Annealing: the temperature goes down so that the primers can stick to the ‘3 side of the DNA segments to copy the base pairs. The temprature is low enough to prevent the DNA to go back to its original shape, and high enough to activate the enzyme that copies the base pairs.
  3. Extension/elongation: the sample gets heated up again, and now the primers will start copying the DNA. When this cycle ends, the amount of DNA has doubled. This cycle will be repeated for about 30 to 40 times, and the number of copied DNA strings will grow exponentially.

I found this photo on Wikipedia that I found really helpful to explain and understand the process:

Schematic drawing of a complete PCR cycle

Gel electrophoresis

When the cycle is completed, it can be tested using the gel electrophoresis technique. This is a method to seperate DNA and other fragments based on their size and charge. The samples are pipetted in wells in the gel, and are placed in a device with two elektrodes that form an electric field. The electric field has a negative charged side and a positive charged side. The negative side pushes the molecules to the positive side. The negative charged molecules tend go move to the positive elektrode faster, and the positive charged molecules move to the negative charged elektrode. The small molecules go through the gel faster, and will reach the end of the gel before the bigger molecules. The test can be stopped when the smallest molecules are at the end of the gel.

The molecules form distinct bands on the gel when placed under UV-light:

gel elektroforese

You can determine the fragment size by using other rows with a sample wich has a known mixture. These known mixtures are often base pair ladders. These fragments all have a specific length (example: 50 bp). These are used to determine the fragment sizes of the samples in other rows, but also to check if the sample went through the gel.

Where do they use it?

  • A biologist or biochemist can use PCR to copy a piece of DNA from a micro organism. The original DNA fragment is then used as example.
  • In a forensic examination, when they find a drop of blood and want to know who it belongs to.
  • When investigating the authenticity of food. They can test the presence of genetically modified organisms.
  • In many infectious diseases PCR can be used to test the presence of a specific pathogen.

And thats it! I hope that I explained it well.

See you in the next post!

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