Kate. Kate. What is it? I've got a riddle for you. I bet you do. No, no, no. You're going to like this one. Okay, hit me. All right. Read me forward and backwards too, I'm still the same word despite what you do. I don't know. Stress spelled backwards is desserts. Yes, but that's not the answer to my particular riddle. But it did remind me that I'm craving cake. And it's almost lunchtime after all. Try again. Okay. Forward, backward, same meaning. Lunchtime. Great. I'm starving. Wait, the answer is noon. Nailed it. Noon is a palindrome. It's the same word backward and forward. So are kayak, madam, racecar, and radar. And check out this sentence. A man, a plan, a canal, Panama. The whole thing is a palindrome. Cool. Now do I get a piece of cake to reward me for answering the riddle correctly? You have a one track mind when you're hungry. No. Your reward is that you get to explain palindromes and why they're important for the molecular scissors we call restriction enzymes. Okay, fine. But after, cake? Deal. But no melon, no lemon. Another palindrome. And we'll learn all about them and restriction enzymes this week on DNA Decoded. Welcome back to DNA Decoded. This week we are talking about all things related to manipulating DNA. In the last video, we learned that DNA cloning involves three crucial steps, cut, paste, and copy. This week we're going to explore how to cut DNA. As we mentioned in the last video, we use molecular scissors to cut DNA. Molecular scissors are actually called restriction enzymes. Restriction enzymes are special kind of proteins, they come in all different flavors. Each flavor recognizes and can cut a specific sequence of DNA. But where do these restriction enzymes come from? And why would a cell have proteins around that could slice and dice DNA? I'm glad you asked. Bacteria actually go out of their way to produce restriction enzymes. Why? Because they defend against viral infections. Humans get viral infections and so do bacteria. Viruses that can attack bacteria are called bacteriophages. A bacteriophage injects its DNA into an unsuspecting bacterium to infect it. As you can imagine, the bacterium doesn't like it very much. So the bacterium's chromosomal DNA contains instructions for making proteins known as restriction enzymes. These enzymes are on the lookout for DNA sequences found only in the bacteriophage. I imagine them as sword-wielding ninjas always ready to protect an emperor or empress from sneaky assassins. When the restriction enzyme ninjas sense the invading bacteriophage DNA, they slice and dice it in a savage display of molecular swordsmanship. That carnage makes the invading DNA useless. Success, assassination averted. Scientists have harnessed those same slicing and dicing powers to cut the segments they need to manipulate DNA. There are hundreds of different restriction enzymes that recognize and cut specific segments of DNA. Restriction enzymes are so vital to DNA research that there are companies dedicated to producing and selling them. They are such a widespread tool that at some universities, you can even buy them out of a vending machine. That's a joke, right? You're telling me I can go and buy restriction enzymes the same way I buy my KitKat? Pretty much, that's how common they are. What do palindromes have to do with all of this? Listen up. This is the cool part. Repeat, cool part alert. Remember, a palindrome is a word or even a whole sentence that reads the same forward and backward. While DNA strands also have DNA palindromes. Here's an example, the strand at the bottom is a palindrome of the strand at the top. Here's another example, most restriction enzymes recognize and cut palindromic DNA sequences. Let's look at a specific example, a restriction enzyme called EcoR1. EcoR1 is a protein that can be used as a restriction enzyme to cut double-stranded DNA at a specific palindromic sequence. By the same token, you could describe me as a human but I could be sub-classified as a professor since my job is to teach. In this image, you can see a DNA helix in orange. The EcoR1 protein seems to be stalking the DNA surrounding and wrapping itself around the double helix. Hello, restriction enzyme and wow, I mean wow. Scientists use X-ray crystallography to figure out the structure of EcoR1 protein, just as they once used X-ray crystallography to discover the double helix of DNA. Remember photo 51? Understanding the structure of a protein helps us to understand its function. This kind of detail is truly awesome but not necessary for our discussion. So let's switch over to a simpler version. I like to call it the blobular version. Blobular is not a word you know. This is coming from somebody who knows the meaning of zymurgy? So here's EcoR1 as a blobular shape. We should rename EcoR1, bob. Blob not bob. And no, the EcoR1 name sticks. Bob is a palindrome and a way better name than EcoR1. A blob of EcoR1 runs along DNA double helix. It's constantly searching for it's specific key, the GAATTC palindrome. Once it finds it, EcoR1 stops and grabs on that specific section of DNA. EcoR1 screeches to a halt at the GAATTC palindrome. Now remember, that the function of EcoR1 and all restriction enzymes is to cut DNA. That's why we call them molecular scissors. EcoR1 targets the GAATTC palindrome and cuts the backbone of the DNA between the G and the A on both strands of the DNA. By now, you might have clued into why restriction enzymes are so attractive for cloning. The cut ends can join up perfectly with the matching cut in another strand of DNA. And of course when it does, the base pairs fits together like a tree because of Watson and Crick rules. Remember, A pairs with T, and C pairs with G. Now in this case, we have a bit of an overhang in the cut. Cut ends that look like this are called sticky ends. They're kind of like Velcro because if they find a fragment of DNA with the right Watson and Crick pairs, they'll snap together. The extremely cool thing is, you don't have to attach them to the same strand you clipped them from. Any strand of DNA with the same kind of matching overhangs will do. Now, let's imagine I used EcoR1 to cut the blue gene from blueberry DNA and made these sticky ends. In reality, we'd use two different restriction enzymes for the job. You'll see why in a minute. In this example, we know that a GAATTC palindrome is on either side of the gene of interest, so we'll use EcoR1 to cut the gene of interest. Then we'll do the same to the plasma DNA. The gene of interest and the plasma DNA have compatible ends. Kate. Yes. I don't know how to tell you this, but it looks like there is more than one way our gene of interest can fit into the plasma DNA. What if it's inserted itself upside down? Okay, I wasn't going to say anything, but yeah, you're right. I didn't want to complicate things too much. But now that you've brought it up, if you use one restriction enzyme to snip both ends of your gene of interest, you'll likely run into problems because the sticky ends could fit together right-side up or upside down. Which means that the base pairs in the gene of interest would be backwards and upside down, which could cause problems in translating the actual genetic code. If the gene is backwards and upside down, the genetic code would be gibberish. Exactly. And the cloned DNA would not produce a functional protein, big fail. To minimize this problem, scientists typically used two restriction enzymes in cloning. They select restriction enzymes that will produce DNA fragments with sticky ends that are incompatible with each other. You want to use restriction enzymes that will snip the gene of interest to fit exactly right in the correct orientation every time. It's important that the scissors you use on the gene of interest make matching shape cuts at the ends of the snip plasma, so things fit together snugly. And this is the power of restriction enzymes. The sticky ends of the gene of interest can attach themselves to the sticky ends of the plasma DNA, just like Velcro. You can combine different pieces of DNA together as long as their sticky ends match up according to Watson and Crick base pairing rules. So take two. This time we'll use two restriction enzymes, EcoR1 and BamH1. While EcoR1 searches for its palindrome, BamH1 searches for its own palindrome. BamH1 cuts both strand of DNA between the two genes. Now, there is no possibility of the sticky ends sticking together the wrong way. The EcoR1 and BamH1 sticky ends can't fit together. They bounce off of each other and float away until they meet up with the sticky ends of the gene of interest. This means that once cut, the sticky ends cannot come back together. Super cool. Now, we can proceed to our next step in the DNA cloning process, pasting together the pieces of DNA.
