Genotyping

Genotyping is a laboratory process that helps determine the genetic makeup of an organism. This method is overall used to find mutations such as insertions and deletions. This knowledge on genetic information could be applied in many different scientific fields such as forensics and biology research. There are many different methods for genotyping but the commonly used one includes PCR testing.

PCR testing is a laboratory technique that is used to amplify small amounts of DNA. In this process, DNA, along with enzymes and primers, is repeatedly cooled and heated. Essentially, the process called thermal cycling is what allows for the DNA segments to be copied exponentially. This consists of three main steps:

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Denaturation: Here, The double stranded DNA is typically heated to 95°C 

to break the hydrogen bonds, separating the strands.

Annealing: In this phase, the mixture is lowered to 50-65°C, allowing primers to bind to their complementary sequences on the single stranded DNA. 

Extension: The temperature is raised back to 72°C and a DNA polymerase enzyme synthesizes a new strand of DNA by extending the primers. This would create new DNA strands complementary to the original template strand.

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The steps start off with the template DNA, which is the sample taken from the organism. This is then mixed with water, Buffer, DNA primers, and a heat stable polymerase. Primers are small pieces of DNA that bind to specific sequences. Once they are binded, the DNA polymerase extends the DNA from the 5’ to the 3’ end. This will essentially create more copies of the DNA strands. After running this mixture through the PCR process, the next step would be gel electrophoresis. To make the gel, it would consist of 1-2% agarose mixed with a buffer. This would have to be heated, mixed until it is homogenized, and then left in the mold to solidify. 

Once the gel has solidified, the mold would be carefully removed, leaving wells in the gel. This gel would need to be submerged into a buffer to run in the electrophoresis chamber. Before turning the electric current on, the samples would need to be pipetted into the wells. After loading the wells with the PCR amplified DNA samples, a DNA ladder would also be added to one of the empty wells in each row to serve as a reference for determining the fragment sizes. 

When the electrophoresis chamber is connected to a power supply, an electric current is run through the gel. Since DNA is negatively charged, it will move towards the positive electrode. Smaller fragments travel faster and farther compared to the larger fragments. So, once the run is complete, the fragments will be in different places, visualized with bands. To see these bands, you would need to put the gel under UV light using a fluorescence viewer. 

Once you put the bands under UV light, you can take a screenshot and transfer it to your computer. Here, it is easiest to determine the genotyping results by comparing it to the DNA ladder. Since the ladder’s bands’ lengths are already known, you can estimate the size of your sample’s fragments. 

These results can be used to determine whether or not the sample has a mutation. We can see if the organism carries a different gene variant. Circling back, this outcome can be useful in many different scenarios. For instance, in research, the genotypes of organisms are crucial to carrying out experiments accurately and effectively. If a wild type organism and mutant organism are supposed to be compared, knowing the genotype is essential. In forensics, genotyping can be used to match the DNA from a crime scene with suspects. And in medicine, genotyping helps identify genetics, which will help predict how a patient may respond under certain circumstances.

[Results put under UV light using a fluorescence viewer.]

[Gel after the electrophoresis run has been completed.]