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Biology: Polymerase Chain Rxn: DNA Amplification

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About this Lesson

  • Type: Video Tutorial
  • Length: 10:20
  • Media: Video/mp4
  • Use: Watch Online & Download
  • Access Period: Unrestricted
  • Download: MP4 (iPod compatible)
  • Size: 110 MB
  • Posted: 07/01/2009

This lesson is part of the following series:

Biology Course (390 lessons, $198.00)
Biology: Biotechnology (16 lessons, $23.76)
Biology: More Techniques in Biotechnology (5 lessons, $8.91)

Many copies of a DNA segment can be made through the process of denaturing the DNA
segment and then polymerizing the new strand using DNA polymerase. In the past this
process was difficult because the heat required to denature DNA would also denature DNA
polymerase.

Thomas Brock found that the DNA polymerase of the bacterium T. aquaticus would not denature at high temperatures. Building on Brock’s findings, Kary Mullis developed the method of DNA amplification known as PCR (polymerase chain reaction). This method overcame the problem of denaturing DNA polymerase at high temperatures by using the DNA polymerase of T. aquaticus. In 1993, Mullis was awarded the Nobel Prize in chemistry for his development of PCR. PCR is a technique that amplifies a segment of DNA into millions, even billions of copies in a short period of time.

Taught by Professor George Wolfe, this lesson was selected from a broader, comprehensive course, Biology. This course and others are available from Thinkwell, Inc. The full course can be found at http://www.thinkwell.com/student/product/biology. The full course covers evolution, ecology, inorganic and organic chemistry, cell biology, respiration, molecular genetics, photosynthesis, biotechnology, cell reproduction, Mendelian genetics and mutation, population genetics and mutation, animal systems and homeostasis, evolution of life on earth, and plant systems and homeostasis.

George Wolfe brings 30+ years of teaching and curriculum writing experience to Thinkwell Biology. His teaching career started in Zaire, Africa where he taught Biology, Chemistry, Political Economics, and Physical Education in the Peace Corps. Since then, he's taught in the Western NY region, spending the last 20 years in the Rochester City School District where he is the Director of the Loudoun Academy of Science. Besides his teaching career, Mr. Wolfe has also been an Emmy-winning television host, fielding live questions for the PBS/WXXI production of Homework Hotline as well as writing and performing in "Football Physics" segments for the Buffalo Bills and the Discover Channel. His contributions to education have been extensive, serving on multiple advisory boards including the Cornell Institute of Physics Teachers, the Cornell Institute of Biology Teachers and the Harvard-Smithsonian Center for Astrophysics SportSmarts curriculum project. He has authored several publications including "The Nasonia Project", a lab series built around the genetics and behaviors of a parasitic wasp. He has received numerous awards throughout his teaching career including the NSTA Presidential Excellence Award, The National Association of Biology Teachers Outstanding Biology Teacher Award for New York State, The Shell Award for Outstanding Science Educator, and was recently inducted in the National Teaching Hall of Fame.

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I have a thought for you. Wouldn't it be great if we could do something with DNA? Wouldn't it be great if I could somehow take that DNA and denature it. Well, you and I know we can do that. Wouldn't it be nice if I could take that DNA and denature it like that and then to that add some polymerase and some nucleotides and maybe I need - remember polymerase won't do anything - so we'll throw some primer in there, too. And if I did that, wouldn't it be nice if I could make two copies of that DNA. So I would have my new nucleotides come in here and add here, and my nucleotides come in here and add there, and come up with this kind of like, DNA, this brand new DNA. If I could just make that happen a little bit faster, I could split those two, and then split the four, and then split the eight, and the sixteen, and the thirty-two, and within hours, I could probably - if you do the calculation - have billions of these.
Well, the sad news is there is a problem with this idea, which would be very cool if I could do it. And what's the problem? Well, here is the problem. The problem is that if you take the DNA and heat it to the point where it denatures - so, we'll go back to my diagram and we're going to do this and we're going to split it into two, here's the problem right here. The problem is, to get DNA to denature, you have to raise that up to 95 degrees Celsius. What do you remember about enzymes? Enzymes, if you recall, are very prone to denaturation. Enzymes are prone to twisting and messing up their active site at like 40 or 41 degrees Celsius. In other words, they twist out of shape and are useless. And DNA polymerase is no exception to this. Yes, this does open up the DNA, but it denatures polymerase.
So, now you're saying, "So, why are you standing here talking to me about this if it can't work?" Well, you know there's more to this story. And here's the story. The story goes back to Thomas Brock. Thomas Brock was studying a group of bacteria that lived in the hot springs of Yellowstone Park. And, the particular bacteria he was studying was called T. aquaticus. The reason that is important, you'll find out in a second. T. aquaticus lived in the hot springs of Yellowstone Park. So hot that they got up to 95 degrees and higher. That's pretty hot. So, that's interesting. And, therefore, their polymerase must be able to withstand hot temperatures. Well, isn't that interesting. So the polymerase of T. aquaticus can withstand hot temperatures. Well, that was Thomas Brock and then in 1985, along came Kerry Mullis, a biochemist who took it to the next step, to the next level. And here is what he realized. He realized if you use the polymerase from the T. aquaticus and he just happened to call it Taq polymerase, that little scenario I described to you a few minutes ago, could work. If we just devise a machine that would heat DNA up and cool it down, and heat it up and cool it down, and heat it up and cool it down in a thermal cycle kind of situation. In fact, let's call the machine a thermocycler. And so what Cary Mullus invented was a process we call PCR. And he won the Nobel Prize for this.
Now, let's talk about PCR and the incredible significance of this and what it has done for biotechnology. I think you can already figure it out. If we can take a miniscule amount of DNA - I'm talking a tiny amount of DNA, a drip of someone's blood with some blood cells in it that have nuclei and somehow get that DNA out of there, we could amplify that DNA, using Taq polymerase. Let's see how.
Here's the principal of what we we're going to call the polymerase chain reaction, otherwise known as PCR. Here we go. Here's how we run PCR. It's very much like that scenario I described to you just a few minutes ago. Here's what we do. We take the small amount of DNA and we put it in a test tube. Little amounts of DNA. We take that DNA and to this DNA, we add Taq polymerase. We add nucleotides, A, G, C and T. We add primer. So what do you need to know? First of all, we need to say "Aha! You gotta know a little bit about the sequence of that DNA." So if you're going to do this societally; if say, for example, you were going to sell a marker that you know occurs in human blood, your biotechnology firm has to know a little bit about at least one of the DNA sequences in that human DNA, so that you can actually make a primer. And this stuff sells for a lot of money. You buy Taq. You buy the primers or you make your own. But you need to know a sequence of DNA that can start out. You can't just throw the DNA in there, because remember, polymerase - what does it need? A primer. That being said, we add these things into there and we're going to heat it up and we're going to heat it to 95 degrees in a box called a thermocycler. In heating it to 95 degrees - now I'm going to go to the molecular level and get out of the test tube level - when you heat it up to 95 degrees, here's what happens. It denatures. When it denatures, the primer comes on. Once the primer comes on, now something else can occur. Now we can get polymerization. Now, after we allow it to cool. This, by the way, after the 95 degrees, we have to let it cool down for about five minutes to let the primer come on, so we're going to cool it to allow the primer to anneal. When that primer anneals, and the DNA polymerases go to work, that's about five minutes. Then, we're going to get our DNA strands. I have a much better picture of this; I'll show you in a second. What do you think we're going to do next? We're going to take that tube and we're going to heat it again. See what's happening here? Of course, the primer is still going to be attached there; that doesn't go away. But you'll see that becomes, after twenty generations or so, the primer becomes a nonentity literally. So, now we're going to separate these and we're going to - 95 degrees, primer will come on this - getting the idea here folks? Primer comes on here.
Let me show you a better picture, but you're getting the idea. Primer here, and now we can go ahead and run this down once again. Watch. Here's a true artist's rendition of this. We start out with my DNA. We open it up. Upon opening it up, we put our primers on. Now that the primers are on, we're going to get the formation of - let's see, what's going to happen? There we go. We have these two. Then, slide this up out of here, because you don't care about that original DNA anymore, because it's literally been subsumed by all of this. Now we're going to get those and now we're going to heat those up one more time. You get the idea what's going to happen? Think. If we go five minutes each time, five minutes, that's twelve, we'll have twelve replications in an hour. We'll have twenty-four in two hours. This thing is going to grow logarithmically. Within hours, you'll have billions of copies of that original DNA. What are you going to use that for? Well, think about it. You have a copy of someone's DNA. If you have a marker, or if you have one way of telling whether it's their DNA or not, just think of the implications of this alone, in the crime field, and criminal detection. Never mind the detection of genes in eukaryotic cells. PCR absolutely revolutionized the way we deal with DNA and the way we make DNA.
Biotechnology
More Techniques in Biotechnology
Polymerase Chain Reaction: DNA Amplification Page [1 of 2]

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