Humans are able to isolate their DNA through a series of different techniques such as using centripetal force and alternating temperatures in order to later use the isolated DNA in a Polymerase Chain Reaction (PCR) experiment. In this article, I am going to discuss how I used my remaining products from my PCR experiment to discover my genotype.
The process I used to deductively discover what my genotype was is called Gel Electrophoresis. Gel Electrophoresis is a method that allows individuals to abstract and analyze a variety of macromolecules depending on their size or charge. In simpler terms, Gel Electrophoresis is used to separate DNA fragments such as DNA, RNA, and proteins based on individual size or charge. First, DNA samples are loaded into small slits at the end of a gel also known as wells or indentations. The gel electrophoresis is utilized by researchers so that they can visually see and analyze the separation of fragments. Furthermore, there is an electrical component that is used in the electrophoresis process, when applied, the electrical current pulls the DNA samples through the gel. Because DNA fragments are negatively charged, they move towards the positive electrode. Lastly, depending on the size and charge of the macromolecules, the molecules will migrate in different directions and at different speeds which help researchers to deduce various conclusions.
Figure 1: Figure 1 illustrates how Gel Electrophoresis results are analyzed. More, there is a graph that represents the relationship between DNA Fragment Size and Distance that were concluded from the results seen at left.
Although Gel Electrophoresis may sound uncommon, it is becoming a commonly used method for medical fields and other scientific experiments. It is most frequently utilized in forensics, molecular biology, genetics, microbiology, and biochemistry. For instance, in forensics, it can be used in DNA fingerprinting. Forensic scientists are able to compare samples of DNA and obtain prints by using this method. After the Gel Electrophoresis lab is completed, it will appear in the bands where it will form a pattern outlining a fingerprint. It could be used in many different ways when testing antibiotics and vaccines. One way they can be tested is for their purity by applying electrophoresis to a solution in the form of a paper strip impregnated with the antibiotic or vaccine. Researchers can differentiate between the antibiotic or vaccine and determine any impurities. They can also determine how concentrated an antibiotic is, which is crucial for applying accurate dosages. Of course, Gel Electrophoresis is not just limited to the things I listed, it can be used in numerous ways. This process has helped people in many ways such as learning more about their genetics and has helped conclude various diagnostics as well.
Figure 2: In Figure 2, an example of an Electrophersis tank, the tool used to separate the DNA fragments, is shown.
In my high school Forensics Science course, we utilized Gel Electrophoresis to analyze the separation of PCR products. Firstly, we began by preparing the agarose gel with SYBR Safe stain. Agarose is a jello-like component that helps separate DNA fragments based on size. In the ends of the gel, where the wells are located our groups filled them with our PCR products from our last lab. Moreover, the gel is produced through a combination of agarose molecules that are held together by hydrogen bonds. In order to prepare the gel for the lab, we had to mix agarose powder with 1X TBE buffer in a 250 mL flask. Then, we had to dissolve the powder by heating the solution to its boiling point. We achieved this by simply microwaving the solution on high for approximately one minute. Once heated, we mixed the solution with 15-second intervals in between until the solution appeared dissolved (the solution should be clear almost like water). After, we had the agarose cool to around 60 degrees celsius by diligently swirling the flask in order to prompt the dissipation of heat. While the agarose cooled, we sealed the ends of the gel-casing tray using rubber end caps, and then placed the “comb” also known as gel cassette in the appropriate notch. Next, we added SYBR Safe, a cyanine dye used as a nucleic acid stain that binds to DNA and absorbs blue light and illuminates green light. Once added to the agarose solution, we mixed them together by swirling the flask. Then, we poured the cooled agarose solution into the prepared gel gasting tray which solidified well after 20 minutes Lastly, we removed the end caps from the comb and were left with our ready to go Agarose Gel Electrophoresis lab.
At this point, my forensics class was ready to begin the separation of PCR products by using Gel Electrophoresis. We placed the gel onto the tray or the electrophoresis chamber and covered the gel with 1X TBE electrophoresis buffer. We used the typical M12 EDVOTEX Model which requires a volume up to 400 mL of buffer. However, in many cases people can also use an MG and M12 Model, a newer model that requires approximately 300 mL of buffer, or an M36 Model which requires at least a volume of 1000 mL. Next, we took our PCR products, our isolated and amplified DNA, and injected 25 µL of it using a P200 micropipette into the wells of electrophoresis tank.
Figure 3: Figure 3 demonstrates the steps to prepare the Gel Electrophoresis tank to be utilized in an experiment.
Once the electrophoresis completed, we removed the gel and casting tray from the electrophoresis chamber. Then we slid the gel off the casting onto a tray. By using a transilluminator, we are able to visually see our separation of PCR product results. The DNA illuminated as bright green bands on the dim background. After observing our results, the class photographed them in order to further analyze them. Lastly, we removed and disposed of the gel and cleaned al of our stations and the equipment carefully.
Figure 4: An example of how the products from my PCR experiment transitioned into being used in the Gel Electrophoresis lab. 1) DNA is extracted 2) Isolation and amplification of DNA 3) DNA added to the gel wells 4) Electric current applied to the gel 5) DNA bands are separated by size 6) DNA bands are stained.
While my class analyzed their individual band of DNA, we learned how to differentiate the different genotypes, heterozygous, and homozygous by analyzing their different individual traits. For instance, those with homozygous DNA will have a thicker band while those who have heterozygous DNA will have a relatively thinner, straight, ad more structural line. By comparing the genotype traits with my sample, it was most similar to homozygous traits. Thus, I was able to infer that my genotype is in fact heterozygous. The reason for my genotype being heterozygous means that both my parents, mother, and father, carry two different DNA strands. Due to the process of child making, I obtained one of the two DNA strands from each parent thus creating who and what I look like today.
Figure 5: This is what a final product of Gel Electrophoresis would look like. These are a set of different DNA bands. By looking at the visual, some of the DNA ladders can be seen with similar lines as a neighboring one. This means that they are similar in their DNA structures.
Edvotek. “Module I: Isolation of DNA from Human Cheek Cells.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Edvotek. “Module II: Amplification of the D1S80 locus.” “VNIR Human DNA Typing Using PCR.” Handout. Forensics I. Pioneer Valley High School. (Nick Enns) 27 Feb. 2020.
Enns, Nick. “PCR Notes.” Forensics I, 24 Feb. 2020. Pioneer Valley High School.
Enns, Nick. “DNA Isolation Notes.” Forensics I, 30 Jan. 2020. Pioneer Valley High School.
Gel electrophoresis. (2020, June 25). Retrieved July 01, 2020, from https://en.wikipedia.org/wiki/Gel_electrophoresis
Gel electrophoresis (article). (n.d.). Retrieved July 01, 2020, from https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/gel-electrophoresis