 Now, I said that I became aware of this respiratory virus and that it was a virus of interest. I became aware in February 2020. In March 2020, it was declared a global pandemic. It first came onto the radar of public health officials in the World Health Organization in December 2019. So a good several months before it came into my brain, scientists were watching what was happening and going, this number one, this virus replicates really fast. Number two, the infection is gnarly. Like it is causing issues with humans and death with humans that we need to watch this thing and we need to control it. One of the most important things to do is to figure out the genome because we pretty much guarantee if we can't figure out the genome, the sequence, then it's going to be hard to develop tests and vaccines. I'm going to show you, I'm going to go back to that website that I showed you earlier and specifically look at this SARS-CoV-2 virus because if you scroll down on this page, they actually have the DNA sequence or the RNA sequence right here. They figured out which parts of the genome code for which proteins and what each of these chunks of the genome actually do. They even have the poly A tail at the end of this mRNA strand, which or this RNA strand, which awesome, right? The ribosome reads it if it has the bling that gets dressed up on. You know what I'm trying to say. The virus has dressed up its RNA so that our cells, our ribosomes will read it. This is such a cool site because it links like the code and where it is in this strand with the actual proteins. Check it out. You can see up there, you can see the proteins, this piece of RNA codes for those spike proteins that allow the virus to get into our cell. Okay, so let's look at, dude, how are we going to sequence this stuff? How do they do that? How'd they figure it out? What I have to tell you is that the process I'm going to describe was discovered in the 70s by a dude named, I can do this, come on now, Frederick. Frederick Sanger in 1977 discovered this process of how to sequence DNA and I'm going to talk about sequencing DNA, but there's one, there's a way that we can go from RNA to DNA. And that's what they did with COVID and that or the SARS-CoV-2 virus and it requires a special enzyme called reverse transcriptase. I love it when words, we can figure it out just looking at this word. So reverse backwards, transcript, that makes me think of RNA, transcription, DNA to RNA, reverse transcription, and then of course, thank you very much for adding the ACE on there, which tells us that that's an enzyme. Okay, nice. So I'm down with reverse transcriptase as an enzyme that I can remember what it's going to do. This was first. So they took reverse transcriptase, added a bunch of nucleotides plus DNA nucleotides. So they took the genetics, the hereditary material out of the SARS-CoV-2 virus, added reverse transcriptase and added DNA nucleotides and voila, ended up with a strand of DNA that matched the RNA transcript. So this is the RNA material found in the virus. That's first. Now we're going to talk about how we sequence the DNA. Here's how it happens. First of all, you got to have a piece of DNA, get enough DNA for four tubes. Interesting. And I'm going to draw you little tubes and I'm drawing them this way on purpose because they're micropiped tubes and I'm not kidding, folks. They're like this big, like maybe what is that, like an inch tall, and they are shaped like this. They're shaped like little ice cream cones. And the amount of DNA enough DNA that you need is like one drop. Like one drop of liquid goes in the bottom of this wild little tube and that's enough. Okay, that's cool. This process that we're going to describe also lets you amplify DNA. So if you have a sample of DNA and you know something about it, you can try primers to bind it and it will let you make jillions and jillions of copies of this process. We're going to talk about that process later in more detail. Okay, so we have enough of the DNA for four tubes. Then we add, we're going to add DNA polymerase. Now just stop a minute and think, why are we going to add DNA polymerase? What did DNA polymerase do when we learned about it before? It's our main DNA replication enzyme. So if we have DNA polymerase in this mix, then we're going to be able to make copies of the DNA. The DNA polymerase will do that job. I'm going to tell you that this is a special DNA polymerase called TAC polymerase and it's a special polymerase found in Archaeans that live in hot springs, thermal hot springs. They first discovered this guy in the Yellowstone National Park in the hot springs and this is significant because it functions at high temperatures. When you do this process, you have to take your DNA, let's see here we'll go purple, you have to denature the DNA with heat and all that means is that you're going to separate it into two strands. You're not going to have a single one piece of DNA, you're going to separate the double helix and you're going to get just two strands which that has to happen in order for replication to happen. That has to happen. Remember this is just the DNA that we created from the RNA found in the virus. Next thing we need to add is we need primers because remember DNA polymerase can't get the job done unless it has a primer that binds to this single strand. So I'm just going to draw my little primer here and once the primer binds, I'm going to not draw it like that, I'm going to draw it down here, you can figure out why that is. Once that primer binds, then DNA polymerase, in this case the hot one, can come in and start making copies of the DNA. Once we have a copy, once we have double-stranded copies made, we can heat it up and cause denaturing and then TAC polymerase can come in and do the job again, make another copy, cool it down so TAC polymerase can do that work, heat it back up, denature it, now we separate those all out again and TAC polymerase can come in and replicate again. You might look at this and be like, okay this is just getting us a whole bunch of copies and it is except there's one other thing that we're going to add. These are chain terminating nucleotides. So hopefully you're looking at this going, okay dude you've got the polymerase and the primers but you also need nucleotides and I'm like, oh right, right, we've got to have nucleotides because we can't add DNA and make copies unless we have nucleotides in the mix. So these guys, these three ingredients are added to every single tube. That's awesome with the DNA and now we've got everything we need except here's the coolest part. Oh my gosh, what color is this going to be? It's going to be yellow because it's my favorite color. We add a chain terminating nucleotide to each tube. So in the first tube we add chain terminating adenines and in the second tube we add chain terminating thymines and then chain terminating guanines and chain terminating nicombritin cytosines. What in the world is a chain terminating nucleotide? Well, this was a really fun lecture to prepare for because I got to look up Legos and I have to find my Lego part picture because this is what I think of when I think of chain terminating things. Nucleotides are like Legos, right? We just can stack them and build them and make giant stacks. A chain terminating nucleotide is like Lego number seven over here. Do you see this? Lego number seven has the bottom part. You can put, have you ever played with those kinds of Legos before? I want to make them into roofs because you're not going to add things on top of the roof. You can just have a flat roof. It's chain terminating because you aren't going to be able to add anything else after that one goes in. The chain terminating nucleotide gets added in to the replication process. The process stops. It's done. And you end up with a fragment of the DNA, a fragment of the DNA that you know ends in A. Dude, how weird is that? So you end up with in, just imagine this for a second. Imagine you get these strands of DNA of varying sizes because everywhere that there is an A it added a chain terminator and it just stopped. Now, there's also regular A's in there. So it's a random draw the dice. It's a roll of the dice. You don't draw dice. You draw cards and you roll dice. And it's random both ways. So DNA polymerase is working in the A tube and pulls, okay, here's a T. I need to add an A, adds the A nucleotide. If it was a chain terminator game over, DNA polymerase is like, well, can't go on from here. Guess I'm going to start a new one and get working on some new ones. If it's a regular nucleotide, then it keeps going and it just is fine. And it ends up making all these fragments in the first tube that end with A. All these fragments in the second tube that end with T. All these fragments in the third tube that end with G. And the fragments in the fourth tube that end with C. Now, dude, you've got a whole bunch of fragments in a drop of liquid. You can't see do-do. What are you going to do-do? Don't do-do because all you have to do is run it through a process, a technique called electrophoresis. I will write that down for you. electrophoresis to the rescue. Seriously. So you take your little drops of DNA that you did a thing with them, but you can't see what you did. And then you run them through this process. This right here is an agar, agar, agar, agarose. That's what it is, an agar, agar. I don't know how. Obviously, I don't know how to pronounce this word, but it's made out of gel. And it's like seaweed gel. And you put little holes, little wells in the ends right here where this guy is putting her, that machine thing is going in. She's dropping the drops of DNA into holes in this gel. And the electrophoresis machine sends an electric charge through the gel. And the electric charge is it's negatively charged by the wells. And it's positively charged at the end. And DNA has a negative charge. So you run, you drop all this negatively charged stuff in the well. You run an electric current through. And the positively charged DNA particles move through the gel. Now, oh my gracious. You can actually visualize where the lines of DNA, like the molecules of DNA, spread themselves out through this gel. And you can imagine if the wells are down here on this end. Actually, it looks like the wells are up here. And I should use. You can kind of see them. I don't know if you can see them, but I can kind of see them right here. And then this is the positive, I mean the negative side, where you put the DNA in. And then you run the current to the positive side. And so all those little particles of DNA are going to move. This is why this lecture was so fun, because I also got to look up, what are they called, jungle gems. Because you know I had to have a visual for this. The DNA molecules going through the gel, little ones are going to go faster than big ones. Now imagine all these tiny humans have to race through this jungle gem. And they have to race like NFL football players. Who's going to win the race through the jungle gem, NFL football players, or the tiny humans? Dude, tiny humans are going to go way faster through the jungle gem than the big NFL players. So you can actually end up concluding that the particles that move farther are smaller. They move farther and faster through the gel, because they're smaller. They're smaller pieces. You can also conclude that we had the A column and the T column. So where the lines come out, we can actually read backwards and determine the sequence of the DNA. Once you know the DNA sequence, you can backward figure out the RNA sequence. This was really long, but we're going to practice. And the next one we're going to look at, okay, what does that actually look like? So we'll have a quick review and then we'll keep going for our other types of biotech stuff related to COVID.