There are some pieces of technology you really
develop an appreciation for. Maybe it’s your phone. Your laptop. Your drawing tablet? But for me, it is the copy machine. Oh, the copy machine. I know it’s not possible, but I could promise you that whenever the copy machine was aware of my presence, that’s when it decided to
malfunction. Especially if it knew I was short on time. And of course when it jams, the fancy copy machines will even tell you what is wrong and how to fix it, but my experience with
trying to follow the instructions of fixing the copy machine makes it worse and then by that by point there’s a line and you feel your heart pounding and you’re embarrassed because now you’ve jammed it for everyone… Yeah, so while I’ve developed an appreciation and respect for the copy machine, I’ve definitely tried to limit my use of it over the years. Saves paper anyway. You may be wondering why is she spending so much time on this? Well, I want to call your attention to a technology– a biotechnology – that works almost like an amazing – and fancy – copy machine. Except not for something on paper. No, it’s for DNA. This biotechnology is PCR. Polymerase Chain Reaction or PCR for short. It provides a way to make more copies of a portion of DNA. Lots and lots and lots of DNA copies, and this technology does not need to happen in a cell either. In fact, it can happen in a test tube. And so you may wonder: #1 How does PCR work? And #2 Why? Why make more copies of some specific portion of DNA? So, let’s briefly answer those two questions. So to answer the first question, the “How
does this work?” question, let’s talk about what we need
first before we get to the steps of how it works. We need the DNA portion that we want to make copies of. We need some kind of buffer to put it in. And then we need things that would be necessary to make more copies of the DNA. So, think about what that might mean from our DNA replication video. We’ll need primers. Recall that primers help DNA polymerase, a building enzyme, know where to go to start its building. We need DNA polymerase, the building enzyme. Fun fact: the DNA polymerase used is often a heat-resistant type of DNA polymerase as PCR uses heat. Typically, the polymerase chosen for the job is Taq polymerase, a type of heat resistant DNA polymerase. Taq polymerase is originally from a type of bacteria that can handle and live in really high temperatures in nature, in hot springs. Pretty cool. We also need DNA nucleotides for the DNA polymerase to build with. So, we can look at the PCR sequence in three major steps. We’ll illustrate with one double stranded
DNA molecule here and let’s assume this is what we want replicated. Step #1 Denaturation. You’ve heard that word “denature” before, right? When we were talking about enzymes? One way we had talked about denaturing enzymes was using heat. And heat is what we will use in this step. This step involves the addition of heat needed to separate the two strands of the DNA molecule. Step #2 Annealing. Ok, so this word means something a little different in biology- basically this is when the two DNA strands that now have been separated by that heat are going to be cooled and be joined by the primers. The temperature for this step should allow the primers to bind to the specific segment of DNA that you want to amplify, which means,
make copies of. Step #3: DNA Synthesis. Remember, synthesis means to make something. With DNA synthesis, we’re going to make more copies of DNA. And to do so, DNA polymerase will begin to
work on both of these strands, and it will use the DNA nucleotides as its building material to amplify the DNA. We should note that the temperature at this step may be a little warmer than the previous step; it needs to be a temperature that is
ideal for the specific DNA polymerase used. Now after finishing one cycle of this, you
have two double-stranded DNA molecules, right? Similar to how it would be in DNA replication within a cell. But you can repeat this now! Except this time you now have two double-stranded DNA molecules to start with. So you repeat with the denaturation, annealing, and DNA synthesis steps. Now you have 4 double stranded DNA molecules. You repeat the steps again. Now you have 8. And if the process is automated by a machine which---why, yes, they do have in science catalogs---you can actually do this fairly
quickly. Which brings you to…why? Why do this? Well any technology that needs copies of a portion of DNA could find PCR useful, but we’ll just mention two examples in our limited time. We mention DNA fingerprinting in our gel electrophoresis video, and we mention that DNA fingerprinting can be a part of a crime scene investigation. Well, in order to have enough copies of DNA samples to run in gel electrophoresis to analyze, PCR can be performed to make copies of the fragments of DNA that are found at a crime scene. Another example: diagnosis of a disease such as one caused by a virus. For an especially relevant example, I can
mention one of the testing types done for the virus that causes COVID-19. COVID-19 is a pandemic that we’re experiencing right now in 2020, and the virus that causes COVID-19 is called SARS-CoV-2. You might have heard of one test type that uses a sample from a nose or throat swab in a “PCR test.” But to be more specific, this test for this
virus is a real-time reverse transcription PCR (rRT-PCR) test. The reason it has this fancier “reverse
transcription” in there is because this virus uses RNA as its genetic material instead of DNA and you have to use an enzyme called reverse transcriptase to convert the RNA into DNA. So, before we can do the regular PCR steps we’ve mentioned, we must convert isolated and purified RNA into DNA. A specific primer is added that will bind
to an area of viral RNA and then reverse transcriptase is used to convert viral RNA into cDNA (complimentary
DNA). Using specific primers and the Taq DNA Polymerase, the cDNA can be copied over and over each cycle in the familiar steps we’ve been mentioning of PCR. See the goal is you need enough copies of
the viral cDNA in order for it to be detectable. The idea being if it’s a positive result,
you have these selective primers binding and you have the Taq DNA polymerase making more
and more copies of the viral cDNA each cycle. In addition, specific fluorescent probes are also used for identification. A certain level is needed for identification of a positive result. And if the virus’s genetic material is not
present in the sample, then primers wouldn’t bind and there would be no cDNA copies produced. If you’d like to learn more about this test
as far as its limitations and also its complexity as we’re being pretty general here, we’ve
included some further reading suggestions in the video details. So those were just two examples of how PCR can be used, but there are many more examples in our suggested further reading links in
the video description. Overall, PCR is such a useful and fascinating technology that will likely remain indispensable for future uses. And it’s a rare day when I pull out that
word “indispensable.” Well, that’s it for the Amoeba Sisters,
and we remind you to stay curious.
