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.
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.