Categories
COVID-19

The Physiology and Anatomy Behind Viruses Ft. COVID-19

Viruses are mainly thought to be infectious diseases that spread easily, originating from a cough or sneeze, or even out of nowhere. Although this is partially true, there is much more behind the development of a virus. Tracing the origins of viruses can be quite difficult because they do not necessarily leave behind any ‘fossils.’ Rather, they only make copies of themselves which means studying their ancestry requires pinpointing the host cell and trying to deduce its origins.

A virus is a non-living microscopic agent that has attachment proteins that act as receptors. Viruses are extremely small, approximately 20 to 400 nanometers in diameter. By comparison, a human red blood cell is about 6,000 to 8,000 nanometers in diameter. The structure of a virus has a center of nucleic acid (either DNA or RNA), and is enclosed in a defensive layer of protein called the capsid. A capsid is composed of protein subunits also known as capsomeres. Its envelope and the cell membrane are also made up of similar material. 

Viruses have either single-stranded DNA, single-stranded RNA, double-stranded DNA, or double-stranded RNA. The type of genetic material found in a particular virus depends on the nature and function of the specific virus. The viral genome can consist of very few genes or up to hundreds of genes depending on the type of virus. The genetic material is not typically exposed but is covered by the capsid in order to be protected from damage.

Scientists have been able to theorize how viruses may develop based on the fact that the genes of many viruses, like those that cause herpes, share similar characteristics with the genes from cells. This theory is known as the Cellular Theory. This theory implies that viruses first started as big pieces of cellular DNA and eventually became independent. Others speculate that viruses came along very early in evolution, and some of their DNA stayed in cells’ genomes. This is known as the Theory of Evolution for Viruses. The fact that viruses infecting humans share similar structural features with viruses could mean that all of these viruses have a common origin, dating back billions of years.

Not all viruses look exactly the same in appearance, but they all share a similar structure. The shape of viruses varies widely. In general, viruses fit usually into two different visual categories. Viruses of humans, animals, and plants are typically spherical and rod-shaped, sometimes consisting of many sides. Viruses of bacteria (bacteriophages) are often shaped almost like a spaceship.

Figure 2: Visual of the structure of a virus that has a “spaceship” appearance.

Because viruses can not exist independently, they must take over a living cell in order to survive and reproduce. When a virus comes into contact with a susceptible host cell, it will latch itself onto the surface using its receptors. The virus will then inject its nucleic acid into the host cell. After, the virus will enter the cell in order to take control of the host by using the protoplasm,the living part of a cell that is surrounded by a plasma membrane, inside of it to create new viruses. Once these new virus particles assemble, they will leave the original host cell and find new host cells and repeat the same process, called the lytic cycle. There is no cell division within the development and replication of viruses. Viruses are only able to be replicated through the chemical synthesis of viral nucleic acid and capsid proteins. In the ending stage of virus replication, host cells that have been attacked may be completely destroyed or suffer little or no harm.

Few viruses go through an additional phase before replication, known as the lysogenic or dormant phase. During this phase, the virus can remain inside the host cell for extended periods of time without causing any changes to the cell. Once activated, however, these viruses can immediately enter into the lytic cycle.

Figure 3: Diagram of a lytic cycle, the process of virus replication.

Viruses cause a number of diseases in the organisms they infect. Human infections and diseases caused by viruses include Ebola fever, chickenpox, influenza, AIDS, and herpes. Plant diseases include mosaic disease, ringspot, leaf curl, and leafroll. Viruses known as bacteriophages cause disease in bacteria and archaeans. Humans can contract viruses through ingestion, sexual transmission, from the air, and many other ways.

Figure 4: Illustration of how airborne diseases can spread.

Vaccines have been effective in preventing some types of viral infections, such as smallpox and the flu. They work by helping the body build an immune system response against these specific viruses. However, it is important not to forget that many viruses can still cause serious damage to living things, and some can actually be fatal. Viruses like COVID-19, are examples of diseases that morphed into a strong and deadly virus that attacks the body and has the ability to kill.

Coronaviruses are a family of viruses that can cause illnesses such as the common cold and it is essentially made up of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). However, the newly discovered coronavirus, COVID-19 or SARS-CO-V-2, became so severe that it caused a worldwide pandemic and has resulted in hundreds of thousands of deaths. Experts say SARS-CoV-2 originated in China and was passed from bats to humans. 

Figure 5: Diagram depicting the human-to-human transmission of coronavirus and its effects on the body.

The name “coronavirus” is derived from Latin: corona, meaning “crown” or “wreath.” Coined by June Almeida and David Tyrrell who studied the human coronavirus, the name was meant to refer to the appearance of the virus. Coronaviruses are large, roughly spherical shaped particles with viral spike peplomers which are actually proteins that lie on the surface of the virus. On average a coronavirus particle has 74 surface spikes all around. The average diameter of a virus particle is around 125 nm (.125 μm). They also contain a positive-sense, single-stranded RNA genome. 

Figure 6: Image depicts the COVID-19 virus and its anatomy.

Inside their envelope lies the nucleocapsid, which is formed from multiple copies of nucleocapsid protein. They are bound to the positive-sense single-stranded RNA genome in a “continuous beads-on-a-string type conformation.” The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell.

Figure 7: Infographic detailing the structure and makeup of a COVID-19 virus particle.

A virus infects the body by entering healthy cells and going through the lytic cycle. There, the infector makes copies of itself and multiplies throughout the body. The new coronavirus would then latch its spiky surface proteins to receptors on healthy cells, especially those in the lungs, and this is why those who have contracted the coronavirus experience respiratory issues. Eventually, the virus kills off some of the healthy cells.

COVID-19 begins with droplets from an infected person’s cough, sneeze, or breath. They could travel through the air or be on a surface that someone may touch before touching the eyes, nose, or mouth. That gives the virus a passage into the mucous membranes and allows it to segway into the throat. Within 2 to 14 days, the immune system may respond with symptoms including a fever, cough, body aches, headaches, shortness of breath, chills, loss of taste, loss of smell, nausea, and other symptoms. The virus moves down into the respiratory tract, the airway that is connected to the mouth, nose, throat, and lungs. Because the lower airways have more ACE2 receptors compared to the respiratory tract, COVID-19 is more likely to travel deeper than the average cold.

Viruses are very complex in the way they manifest, and there is still much research that has yet to be discovered about viral infections. Doctors are continuously working close with researchers to develop stronger and more efficient ways of combating these infections. Currently, there are different vaccines being made in order to eventually fight COVID-19 virus cases.

Figure 8: A visual referencing the symptoms that may come with COVID-19.

Citations

“2-13. MORPHOLOGY AND PHYSIOLOGY OF VIRUSES “. Nursing411.Org, 2020, http://nursing411.org/Courses/MD0151_Principals_Epidem_Micro/2-13_Principals_Epidem_Micro.html. Accessed 20 Aug 2020.

“Bacteriophage Life Cycle Animation”. Thoughtco, 2020, https://www.thoughtco.com/bacteriophage-life-cycle-animation-373884. Accessed 1 Sept 2020.

Baines, Luara. “Virus Notes.” Physiology/Anatomy, 30 Aug 2020. Pioneer Valley High School.

“Coronavirus”. En.Wikipedia.Org, 2020, https://en.wikipedia.org/wiki/Coronavirus. Accessed 1 Sept 2020.

“Coronavirus Disease 2019 (COVID-19) – Symptoms And Causes”. Mayo Clinic, 2020, https://www.mayoclinic.org/diseases-conditions/coronavirus/symptoms-causes/syc-20479963. Accessed 1 Sept 2020.

“Learn How Virus Replication Occurs With This Detailed Primer”. Thoughtco, 2020, https://www.thoughtco.com/virus-replication-373889. Accessed 20 Aug 2020.

Alexis Dehorta, Youth Medical Journal 2020

Categories
Biomedical Research

Discovering Genotypes Through Gel Electrophoresis

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.

Works Cited

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

Categories
Biomedical Research

High School Students Replicate their DNA During a PCR Lab Experiment

I learned about PCR in my junior year in my Forensics course. As the day came closer where we conducted a lab using PCR, I came to realize how difficult and complex this lab was going to be. Before learning the concepts of PCR, I had questions such as “what is PCR, and how does that relate to replicating one’s DNA?” “What is PCR used for, and what is its relevance in the world?” Questions like these had me on the edge of my seat, eager to learn more about it. 

Polymerase Chain Reaction, or PCR, is a process that allows an individual to replicate many copies of a precise segment of their DNA through a series of calculated procedures. Ever since it was first invented in 1984 by Dr. Kary Mullis, It has become revolutionary in the science of DNA fingerprinting due to its feasibility to amplify DNA. To begin replicating DNA, there needs to be a sample of DNA to use. A DNA sample can be obtained in many ways: blood, semen, feces, hair, saliva, tissue, and cells. For our high school-based experiment, our class used the DNA within our saliva. Moreover, Taq Polymerase is fundamental to the experiment because it helps in the DNA replicating process. Although DNA polymerase is typically utilized in PCR, our class used Taq polymerase because it can withstand much higher temperatures and denaturing conditions. It is also much easier to isolate. Taq DNA Polymerase is a naturally occurring set of proteins that copy a cell’s DNA before dividing itself into two. When the DNA Polymerase finds itself bumping into a primer that is base-paired with a longer piece of DNA, it will attach itself to the end of the primer and then add nucleotides. Because primers are mostly man-made and created within a laboratory, they can have as many nucleotides, molecules consisting of a nucleoside and a phosphate group, as one wishes. 

FIGURE 1: A representation of DNA Polymerase function in PCR.

Now the question that still stands is, “What is its relevance in the world?” PCR can be used for a variety of purposes, but it is most frequently used in Forensics analyses and Medical diagnostics. By examining several different STRS from a particular individual, examiners can obtain a unique trait that is unlike anyone else’s, and they will be able to use that information to determine many different factors of that person. For instance, in Forensics, PCR can be used to match a suspect’s DNA to one that was found at a crime scene. Forensic scientists could take the DNA sample found at the crime scene and from suspects of the crime, replicate the DNA samples using the PCR process, and compare and contrast the samples by examining highly polymorphic DNA regions. They then would be able to determine who may have been responsible for the crime. Further, many specialists use the PCR process every day to diagnose individuals with a disease. Through PCR, specialists can examine any inconsistencies or mutations in the replicated DNA sequences, and determine whether or not a person has a particular disease. They can also use it to identify bacteria or viruses.

To begin the experiment, we had to extract our DNA from our saliva. We did this by rinsing our mouths with saline, salt water, and spitting it into a cup. The saline solution helps neutralize the DNA charge and remove proteins that are potentially bound to the DNA. This allowed us to have pure DNA to use for the experiment. Now that we had our pure DNA, we had to isolate the DNA from the human cheek cells. It is important to have freshly isolated DNA because it will provide pristine amplification results compared to much older and degraded pieces of DNA. To begin the isolation process, my classmates and I took our DNA solution and transferred 1.5 mL of DNA solution into a small tube. To transfer our solutions, we used a p1000 micropipette to extract a precise amount of DNA solution from the cup and transferred the amount into the tube. The reason we transferred our solution into a small tube is so that it could fit into a centrifuge. The centrifuge spins the solution at high speeds for approximately two minutes to separate the components of the solution with centripetal force. 

After the class completed their first centrifuge stage, they took their tubes containing the potentially separated solution and resuspended the cheek cells in a 140 µL lysis buffer, a solution used to break open cells. This was accomplished by pipetting up and down with a p200 micropipette and by vortexing vigorously. Next, we placed our tubes into a water bath float. We incubated the tubes containing the solutions in a 55 degrees Celsius water bath for 5 minutes. After 5 minutes, we took our tubes and flicked the tube vigorously for about 20 seconds. 

We then put the tubes in the bath to incubate again in 99 degrees celsius water for 5 minutes instead. After the 5 minutes, we placed them into the centrifuge once more for 2 minutes at full speed. Lastly, we took our tubes and distributed 80 µl of supernatant using a p20 micropipette into a separate tube. Now that our DNA was successfully isolated, it was time to move onto the amplification stage in the PCR process.

We began the next stage by taking a fresh 0.2 mL PCR tube and added a 20 µL D1S80 primer mix (yellow), 5 µL extracted DNA (red), and a PCR EdvoBead PLUS which provides reagents for approximately 25 PCR reactions. PCR Beads have been optimized for PCR reactions and contain buffer, nucleotides, and Taq DNA Polymerase.  Last, we mixed together our solutions. Our final mixture was a beautiful light orange color, and now that we finally had our solution that contains positive control primers, template DNA, and PCR components, we were ready to begin the PCR amplification of our DNA.

Figure 2: A visual representation of the steps to isolate DNA.

There are three major steps in replicating DNA in the PCR process. Step one of the PCR process is denaturation. Breaking open of the cells or denaturing provides a single-stranded template for the subsequent step. To break open the cells, the tube is heated at 94 degrees celsius for 15 seconds. The initial denaturation will start at 94 degrees celsius for 30 seconds instead. Step two in the process is annealing. Also known as the cooling process, annealing allows the reaction to cool at 65 degrees celsius for 30 seconds, so the primers can bind to the complementary sequences on the single-stranded template DNA. The last major step in PCR is an extension, sometimes referred to as elongation. In this process, the reaction is placed in raised temperatures of 72 degrees celsius for 40 seconds. Each step of the process will go through 32 cycles repeating these steps chronologically over a course of 2 hours. Lastly, the solution will sit for its final extension at 72 degrees celsius for 30 seconds. After we had finished amplifying the DNA, we had over a million copies of specific DNA sequences in the palms of our hands. Although this may seem like an enormous amount of DNA, it can hardly be seen in the solution.

Figure 3: Illustration of the three major steps in PCR: denaturation, annealing, and elongation.

Now that we have completed the PCR process, we were able to use our DNA separations for another lab. In the next article, I will explain how I used my separation PCR products in Gel Electrophoresis and was able to determine my genotype.

Works Cited

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.