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Dec 6, 2023

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Kaden Miller Bio-181 Lab TR-100 3/21/23 Professor Lauchnor Application of Molecular Biology Introduction The process of DNA extraction is used to separate DNA from cell nuclei. Finding out if the DNA has a disease is the goal of the DNA extraction. Breaking apart the cells to release the DNA is the first stage in the DNA extraction procedure. By lysis, the cell was rupturing open. When an osmotic imbalance causes too much water to enter a cell, the result is lysis, which causes the cell to rupture. Separating DNA from proteins and other cellular entities is the second stage. A sodium solution is added to separate them, allowing the proteins to be freed from the DNA. As much protein and cellular waste must be eliminated in order to obtain a clean sample of DNA. Precipitating the DNA with an alcohol compound is the third step. This process is crucial because it prevents the DNA and proteins from precipitating together in the alcohol by keeping the proteins dispersed in the aqueous layer. The DNA precipitates in the presence of ethanol or isopropyl alcohol. DNA tends to cluster together when it exits the solution, making it visible. Finally, the DNA must be cleaned and examined to see whether it contains any illnesses that have hereditary origins. Another method to demonstrate the presence of DNA in the materials is gel electrophoresis. When examining DNA, there are two main things that happen. A method for producing several copies of a certain DNA is called polymerase chain reaction, or P

PCR.PCR is a process that involves three key phases. Denaturation is the first step that has to be taken. Breaking hydrogen bonds, or denaturation, enables the separation of the double helix strands. The annealing of the mixture is the next stage. As a result, the mixture can cool. The cooling mixture makes it possible for the primers to bind to certain DNA sequences. Extension is the third and last phase. The enzymes can stretch the primers and start to generate new DNA when the temperature is raised again. Taq polymerase is the enzyme that is frequently utilized in PCR. It is the most widely utilized enzyme in the PCR process and is used to automate the repeated processes in this crucial technology for amplifying certain DNA sequences. Because Taq DNA catalyzes and integrates nucleotides into the DNA that is being duplicated, it can also be referred to as a primer. The gel electrophoresis is a crucial part of the DNA analysis process. By using the process of gel electrophoresis, little pieces of DNA are drawn by an electric current across a gel matrix. Additionally, it enables the size-based separation of DNA fragments. Gel electrophoresis is an important method that is frequently used to separate and study DNA segments. The DNA fragments will glow when exposed to UV light after being dyed with a DNA-binding dye, making it possible to observe the DNA present at various points along the length of the gel. Alu elements are among the most crucial factors in DNA analysis. Genes that are particular to a tissue are regulated by Alu elements. They can occasionally alter how a gene is expressed and are implicated in the transcription of adjacent genes. The plasminogen activator gene is also known as TPA-25, an element that is included inside the intron. Within the procedure, TPA-25 is increased.Additionally, the presence or lack of TPA-25's Alu insertion affects how the product amplifies. Students will extract the DNA in the lab and use it as a template in PCR to check for the Alu element. P

Data Figure 1. Gel 1. Presence of GMO Gene in Papaya, second food, third food, and fourth food. Table 1: Gel 1 lane legend and results LANE 1 2 3 4 5 6 7 8 9 10 SAMPL E ladder NT 1 Papaya Organic Tomatoes GMO Plant GMO Plant GMO Plant GMO Plant Results: + - - - - - NT = no DNA template, negative control GMO primer = indicates presence of a promoter for a transgene Plant primer = chloroplast gene P

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Figure 2. Gel 2. Presence of GMO Gene in Papaya, second food, third food, and fourth food. Table 2: Gel 2 lane legend and results LANE 1 2 3 4 5 6 7 8 9 10 SAMPL ladder NT 2 Corn Organic Apples Yellow Papaya P

E Squash GMO Plant GMO Plant GMO Plant GMO Plant Results: + - - + - - - - - - NT = no DNA template, negative control GMO primer = indicates presence of a promoter for a transgene Plant primer = chloroplast gene Figure 3. Gel 3. Presence of GMO Gene in Papaya, second food, third food, and fourth food. Table 3: Gel 3 lane legend and results P

LANE 1 2 3 4 5 6 7 8 9 10 SAMPL E ladder NT 3 Papaya Potato Chip Tofu Tomato GMO Plant GMO Plant GMO Plant GMO Plant Results: + - - - - - + + - - NT = no DNA template, negative control GMO primer = indicates presence of a promoter for a transgene Plant primer = chloroplast gene Methodology Typically, a number of crucial stages are included in the approach for DNA extraction from food samples in gel electrophoresis. The food sample is first procured and processed to get an appropriate quantity of DNA. In order to release the DNA, the sample may need to be ground or homogenized. The DNA is then purified and concentrated using a commercial DNA extraction kit or another appropriate technique. After the DNA has been extracted, it is put through a process called gel electrophoresis, which divides and examines DNA pieces according to their size and charge. In order to do this, the DNA sample must be loaded into a gel matrix, which is commonly constructed of agarose or polyacrylamide. The DNA fragments must then be driven through the gel by an electric current. Analysis The process of amplifying a segment of DNA into a million copies using DNA polymerase is known as a polymerase chain reaction, or PCR. PCR is useful because it enables the DNA to be amplified into millions of copies to provide a lot of samples for genetic material analysis and investigation. Denaturation, annealing, and extension are the three cycles that make up the PCR cycle. At a high temperature (between 95 and 98 °C), denaturation enables the hydrogen bonds to rupture, leading to the separation of the two strands. Primer binding to the DNA template at either end of the sequence is made possible by annealing, which is the process of chilling a P

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mixture at a lower temperature (55–65°C). The temperature is rising (to 72 °C) due to extension. The enzymes may now bind to the primer and start constructing new DNA strands as the temperature rises. The goal of the entire process is to create a copy of the original DNA, which comprises molecules from both the old and new DNA strands. The foods that were shown were corn in gel two for the plant and in gel three tofu was shown in both plant and GMO. If a sample displays no visible band, the sequence is proceeding correctly, and the band will eventually disappear. When the template DNA is merely paused, this typically occurs. An exceptionally stable secondary structure could potentially be to blame for this. Eight students failed to successfully amplify the DNA. An unsteady structure may be to blame for this. The number of students in the class, the length of the lab sessions, and the equipment's accessibility are all elements that must be taken into account while organizing and carrying out this experiment. As for the lanes that did not show there are many answers to why that is. Failure of the PCR reaction might have been brought about by improper reagent preparation, poor reaction conditions, or problems with the thermocycler. There would be no DNA to amplify if the PCR process failed, and there would be no visible bands on the gel. Poor quality or amount of template DNA: The quality or quantity of the template DNA utilized in the PCR reaction may be subpar. A poor yield or damaged DNA that cannot be amplified might be the consequence of DNA degradation or improper DNA extraction. The primers used in the PCR reaction may not be able to anneal to the template DNA or they may amplify the incorrect DNA fragment if they were designed incorrectly. This can result in unexpected bands or a lack of loudness. Contamination: Lack of amplification may be caused by contamination during the PCR process or gel electrophoresis. Reagents, machinery, and environmental conditions are only a few examples of the many sources of contamination. Error in gel electrophoresis: Bands may not develop on the gel or be visible if P

there is an issue with the gel electrophoresis procedure itself, such as improper loading of the gel, uneven gel, or inadequate voltage. Conclusion To enable vision during electrophoresis, the sample (DNA, RNA, or protein) is extracted, purified, and combined with a loading solution that contains a tracking dye. When making agarose gel, buffer solution and agarose powder are combined, heated, and then poured into a gel mold using a comb. The gel is put in an electrophoresis tank that is filled with buffer solution after the comb has been removed and the gel has solidified.Using a micropipette, the material is put into the wells on the gel. To ascertain the size of the fragments under analysis, DNA size markers are also put into a single well. The DNA or protein molecules migrate across the gel matrix according to their size and charge when an electric current is introduced to the gel. Larger molecules travel more slowly across the gel than smaller ones do. To see the DNA or protein bands under UV light after electrophoresis, the gel is dyed with a fluorescent dye (such as ethidium bromide). The size and number of DNA or protein fragments in the sample are estimated from the gel picture. Imaging software or a comparison of the sample bands to size markers of known sizes can be used to do this. The data showed that only corn (plant) and tofu (plant and GMO) were shown during the end of the experiment. Understanding the polymerase chain reaction and its processes, as well as the amplification of a DNA sample using gel electrophoresis, were the learning objectives for this lab. P

References Areda, D., Boyles, R., Francis, G., & Hite, A. (2016). Laboratory manual for General Biology I. Retrieved 13 February 2021 from http://lc.gcumedia.com/bio181l/laboratory-manualfor-general-biology- i/v1.1/#/chapter/6 Gupta, N. (2019). DNA Extraction and Polymerase Chain Reaction. Journal of cytology. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425773/ Reece, J. B., Urry, L. A., Cain, M. L., Wasserman, S. A., Minorsky, P. V., Jackson, R., & Campbell, N. A. (2011). Campbell Biology. Retrieved 13 February 2021, from https://viewer.gcu.edu/24WWXP P

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