Polymerase Chain Reaction

by Georgina Cornwall, PhD

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    00:00 Now let us move on to how we could amplify things much more easily. As I said before, previously we had only bacterial cloning in order to amplify DNA. Now when you think about how long it might take even though bacteria reproduced fairly quickly, it could take a long time to get a lot of copies of DNA. PCR machines really up the ante on our ability to create a lot of copies of DNA, in fact, an infinite number of copies of DNA of interest. What we are doing with the PCR machine is replicating DNA in vitro, so in a test tube. We take the DNA that we are interested in and we will heat it up. This is how the process works. We heat it up and separate the DNA and then we ask the DNA to replicate. We cool it down, primers will attach to it and the DNA will become replicated. And then we can cool it off and heat it up again. Now the key is this heating to separate the two DNA strands. It is hard enough to separate the hydrogen zipper down the middle. So we can have two pieces of DNA upon which replication can work.

    01:27 Then we need to somehow attach primers. Well if we keep it hot, then the primers can't stick so we cool it down. So when we add primers to the test tube, the primers will stick to the DNA. We are going to add a DNA polymerase. The DNA polymerase works at a particular temperature, but it might be too warm when we heat it up for the next cycle because this is cyclical. We are going to ask it to polymerize them. We are going to heat it up and separate the DNA strands.

    02:00 We need a DNA polymerase that can withstand heating. Somebody found DNA polymerase, great idea in bacteria that inhabit hot springs in Yellowstone National Park. It is called thermus aquaticus bacteria and we call it Taq polymerase. We can then use Taq polymerase, which will polymerize or add nucletides to DNA sequence at a much higher temperature than normal and so we can allow DNA polymerization to happen in these heating and cooling cycles whilte DNA polymerase can be maintained in the heating. So essentially we split DNA, replicate it, and anneals. Then we split DNA, replicated it, and anneals. Every round in the PCR machine will double the amount of DNA. In this sense, we can have an infinite number of DNA molecules created, which is really cool because now we can take a very small amount of DNA, for example from a crime scene and amplify it infinitely, so we can run DNA fingerprints.

    03:07 We can re-run the prints. Previously if we only had a little bit of DNA, you couldn't do much testing. You might have one shot and if they asked you to replicate the test, it wasn't possible because you were out of DNA. So PCR has really changed a lot of fronts especially on DNA fingerprinting, but also in DNA analysis if we were looking at embryonic cells and trying to determine whether an embryo has predisposition to certain conditions.

    03:39 The DNA can be sampled from a cell or one of the cells in that and it can be amplified.

    03:46 We don't have to take a huge tissue sample to see if DNA contains any of the particular markers asociated with cancers and such. PCR is really a groundbreaking technology in genome exploration. In this lecture, we have covered quite a lot of stuff at the foundation of biotechnology. First of all, you should probably be able to explain how restriction enzymes work to produce recombinant DNA as well as being able to discuss the role of gel electrophoresis in biotechnology. We are going to see a lot more of that in the future. You should also be able to explain the role of vectors in molecular cloning and you should be able to describe the process of PCR and how that has improved our access to techniques in biotechnology. Thank you so much for listening. It has been a pleasure. I will see you in the next lecture.

    About the Lecture

    The lecture Polymerase Chain Reaction by Georgina Cornwall, PhD is from the course Biotechnology.

    Included Quiz Questions

    1. Synthesis
    2. Renaturation
    3. Denaturation
    4. Annealing of primers
    5. Reverse transcription
    1. Exonuclease
    2. DNA template
    3. Taq DNA polymerase
    4. Primers
    5. dNTPs
    1. Amplification of DNA
    2. Amplification of RNA
    3. Amplification of protein molecules
    4. Synthesis of lipid bilayers
    5. Synthesis of an artificial 30s ribosomal subunit

    Author of lecture Polymerase Chain Reaction

     Georgina Cornwall, PhD

    Georgina Cornwall, PhD

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