[Music] [Announcer:] And now, here's everyone's favorite chef: Felicia! [Felicia:] Hello. Welcome to my kitchen and to Felicia's Fabulous DNA Eats. Today, I'm going to show you how to make DNA pasta for thousands. We begin with a single little strand of DNA. You can't see it in this pot of water but, trust me, it is in there. [Caitlin:] Felicia, for the love of Watson and Crick! What are you up to? [Felicia:] Shh! I am in the middle of my brand new show. Stand back! I'm about to make lots and lots of DNA. [Caitlin:] You can't start a Polymerase Chain Reaction in the kitchen. Come on, let's get you to a proper lab, chef. After all, this is... [Both:] DNA Decoded! [Music] [Caitlin:] Much better. Now, lose the chef hat and put on your lab coat. We've got a murder to solve. In this video, we're going to show you a technique scientists use all the time to play around with DNA in the lab. Forensic scientists use it to bust criminals, archaeologists use it to study 4000-year-old mummies, and researchers use it to study the bubonic plague. [Felicia:] The technique is called Polymerase Chain Reaction or PCR. PCR will make copies of the suspect's DNA so we can solve our murder mystery. It's a technique used in biochemistry labs all the time. It's revolutionized the field of molecular biology, because it really is like a cooking show. If you use the right ingredients and the right techniques, it should work every time. Meet my sous-chef for today, Daniel, one of my biochemistry students. [Caitlin:] Thanks for helping us out today, Daniel. [Daniel:] My pleasure. [Felicia:] All right, let's begin. [Caitlin:] Okay, first things first. Safety! We need to make sure that we're all wearing our personal protective equipment. For this lab, we need a lab coat and some gloves. Next, we need to prepare our equipment. We need tubes, a tube rack, micropipettes, micropipette tips, a disposal bin, an ice bucket, and a thermocycler. [Felicia:] And now for our ingredients -- [Caitlin:] -- you mean reagents. [Felicia:] You are no fun! Our list of ingredients (or reagents) is outlined in this checklist. Just like a recipe, the reagents are listed along with how much we'll need of each. [Caitlin:] Our key reagents are water, buffer, template DNA, primers, Taq DNA polymerase, and dNTPs. Just like cooking, it's very important that we add in all our ingredients in the right order. Otherwise, when we get to the end, things won't be quite right. Okay. So, let's start by mixing together our super mix. What's in the super mix? [Caitlin:] Water, the solvent of life. [Felicia:] Buffer, that's a mix of salts and such to help the reaction along. [Caitlin:] Our next ingredient is template DNA. This is the segment of DNA that we're interested in studying. We'll use PCR to make multiple copies of it. [Felicia:] Next are primers. Primers are the short segments of single-stranded DNA that are used to mark the spot where we want to start copying the strand of DNA. Because DNA is a double helix that runs in opposite directions, we'll need to select two primers. One for the starting point on each strand of DNA. [Caitlin:] Primers come in a variety of flavours. You have to choose primers that contain complementary base pairs for your starting points. Our secret ingredient: Taq DNA polymerase. You can think of Taq DNA polymerase as a master bricklayer. She finds the primer's starting point on the template DNA and then selects complementary nucleotides to build a new strand of DNA. [Felicia:] Which brings us to our last ingredient... nucleotides! Remember, a nucleotide is a nucleobase with the sugar and phosphate attached. Nucleotides are the building blocks of DNA. So, we'll need them if we want to build copies of our template DNA. Remember to change your tip when you switch to a new ingredient. Otherwise, you can contaminate your reaction and your reagent. Now, for the most exciting part of the entire protocol. We throw our template DNA in our handy dandy thermocycler. It's a cool gadget that'll do all the work for us. It's kind of like a slow cooker. [Caitlin:] This machine is a lifesaver. You put in the test tube, program the three steps ahead of time, set the repeat for 30 cycles, closed the lid, start the program, and walk away. [Felicia:] Step one. We want to denature the template DNA. This means that double helix of the template DNA will be unzipped into two single strands of DNA. All we need to do is apply a LOT of heat. When we kick up the temperature to 95 degrees Celsius (204 degrees Fahrenheit), the noncovalent bonds between the base pairs come apart and voila, we go from a double helix to two single strands. Each strand will serve as a template for the creation of a new DNA double helix. [Caitlin:] To give you an idea of how hot this is, remember that water boils at 100 degrees Celsius (or 212 degrees Fahrenheit). So, almost but not quite at that temperature. [Felicia:] Step two. Okay, so now that we have two single strands of DNA, the thermocycler turns the heat down a bit. We're going to knock it down to about 62 degrees Celsius, (about 140 degrees Fahrenheit). [Caitlin:] Why are you doing that?! Won't decreasing the temperature bring the strands of template DNA back together? [Felicia:] Well, no... Remember, I sprinkled DNA primers in our mix. We're giving the primers an opportunity to sit down on the DNA to start the process of making a new copy. [Caitlin:] And voila, we have a PCR reaction! [Felicia:] Actually, that would be a Polymerase Chain Reaction reaction. It's just PCR. [Caitlin:] We'll turn up the heat again - not by much though, say 72 degrees Celsius -- [Felicia:] and step back and let Taq DNA polymerase do its work. [Caitlin:] You might remember Taq DNA polymerase from our DNA replication video. [Felicia:] The Taq DNA polymerase will find the location of the DNA primer and begin adding nucleotides one at a time according to Watson-Crick's rules: A bonds with T and C bonds with G. It uses the ingredients in the buffer to bond the nucleotides to the strands of the template DNA. The thermocycler will repeat these steps 30 times, cycling between 95, 62, and 72 degrees until they are over a billion copies of our original strand of DNA. [Caitlin:] Here's what the whole process looks like, from beginning to end. [Caitlin:] Gotta love the thermocycler! You set the cycles, put your tube in, press start and voila, DNA for days! [Felicia:] Once we've cooked up some copies of the DNA, we can compare the blood from the crime scene with the samples from our suspects. Come on Caitlin, let's go already. All this cooking is making me hungry. [Caitlin:] Pasta? [Felicia:] Burgers. [Caitlin:] Felicia and I, and our wonderful student, have given you a real life demonstration of the technique known as PCR. Now, after watching our "cooking show," you might come away mildly amused -- we hope -- perhaps a bit interested in this PCR thing, and even driven to understand it a bit better. But, what I want to highlight here is the immense power of this simple technique. There is no question that without the invention of PCR, we would not have been able to achieve the same level of advances in the field of biotechnology and biomedical sciences. We would not have DNA-based CSI evidence techniques. There would be no human genome project, no personalized medicine, no genetic testing, and no easy way to manipulate DNA. Caitlin out.
