Polymerase Chain Reaction (PCR) is a very important lab test that can be used to detect genetic material and has many use cases, like forensics in criminal investigation and virus/bacteria detection.
PCR has been very useful in the fight against SARS-CoV-2, as it is used for easy and reliable testing and diagnosis, which can be used for potential isolation and contact tracing.

Goal:

This article will show you how to perform PCR, which can then be used to identify a certain person or virus/bacteria.
In this article, I will perform PCR using a strand of hair to test for a person’s genotype of the tPA-25 gene.

Background Info (Theory):

PCR is the process of copying/amplifying a specific section of DNA, which will then give you more DNA to test on, so you only need a small sample to get a result.
The idea behind PCR is to mimic the DNA replication process many times, by using the simple building blocks of DNA and the enzymes involved.
The DNA sample is heated up enough so that the hydrogen bonds between the two strands fail and the DNA unzips.
Then nucleotides, DNA polymerase I, and DNA primers are added.
The DNA primers will bind to the DNA sample for the specific gene and then the DNA polymerase will bring the nucleotides over to the DNA primer to start producing more of the gene.
Then this process is repeated many times up to factors of 20-30 to get a big enough sample to test on.
However, normal DNA polymerase would denature at high temperatures, but a heat-resistant DNA polymerase called Taq Polymerase has been found in bacteria that live in geysers and have adapted to the heat, so we will be using that.
So in summary:
Denaturation: The DNA sample unwinds under the heat (95 C).
Annealing: Primers bond to the specific sequence after some cooling (55-65 C).
Extension: DNA Polymerase I builds the strand from added nucleotides (72 C).

The Procedure:

Step 1 (Preperation):

Before doing anything, you should have safety goggles and gloves on, as PCR entails the use of very hot liquids and different chemicals.
When you are ready, you should find your DNA sample and all other materials needed.
Put your DNA sample into a small 1.5mL tube that contains 10% Chelex solution, and mix to extract the DNA into the solution.
Be sure that the Chelex solution is all over the DNA sample and that it isn’t stuck in the bottom of the tube.

Step 2 (DNA Extraction):

For DNA extraction but your DNA-Chelex tube into a 58 C water bath for about 10 minutes, but shake the tube at the halfway point.
Then take the tube out and shake it again and then put it in a 100 C water bath for about 5 minutes.

Then take the tube out and put it in a 6000xg centrifuge for about 7 minutes for thorough mixing.
After that let the tube refrigerate for about 15 minutes and then put it back in the 6000xg centrifuge and let it sit for 5 minutes.

Step 3 (Denaturation, Annealing, and Extension):

For the actual PCR process, you only need to add the components and a machine will do the rest.
First, you need to add the free nucleotides, the primes, and the DNA Polymerase, this should be combined in a tube, and you only need to add about 25 microliters.
After adding the required components shake the tube again to mix and set aside.
I would always recommend having a control, so you should ideally have another tube that you know will have the gene, if you are to do any tests.
Then use a Thermocycler, that will periodically increase and decrease the temperature over about a day.
This can technically be done by hand, but using a Thermocycler is easier, and in the meantime, you can prepare the experiments you want to run on the gene, like a gel.

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