Polymerase Chain Reaction (PCR) in Pigs

Don Lee, Presenter Author

12/18/2018 Added

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Polymerase chain reaction (PCR) is a technique that allows you to make millions or even billions of copies of sections of DNA. This video walks you through how it works.

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[00:00:12.189]Polymerase Chain Reaction,
[00:00:13.960]or PCR as it's often called,
[00:00:16.750]is an incredibly powerful DNA analysis technique
[00:00:20.670]that is based on the process of DNA replication.
[00:00:26.600]So, I'm gonna give a little narration of this
[00:00:29.760]animation developed by a previous USDA project
[00:00:34.760]at University of Nebraska,
[00:00:36.700]and let's see if we can understand how PCR works.
[00:00:39.570]So, we're gonna start with getting at the DNA
[00:00:43.053]that is part of chromosomes that are inside the cells,
[00:00:49.463]that make up tissues that we sample.
[00:00:55.205]so the key to PCR is that
[00:00:59.050]we're gonna make copies of just a specific segment of DNA
[00:01:03.780]from all the DNA that we extracted out of our tissue.
[00:01:08.940]You're gonna see how we can be very specific about that.
[00:01:12.620]Now, before we can do PCR
[00:01:15.420]we have to obtain our DNA sample,
[00:01:17.950]and then we have to do a little shopping.
[00:01:19.820]We have to buy an enzyme called Taq polymerase,
[00:01:23.920]we have to buy the individual DNA nucleotides
[00:01:26.990]that will be polymerized into new copies of DNA.
[00:01:32.590]We need to buy what's called a DNA primer,
[00:01:35.130]you'll see where that will fit in,
[00:01:36.570]and we have to buy some test tubes and reaction buffers.
[00:01:41.700]Okay.
[00:01:43.150]Our lab is well stocked,
[00:01:44.580]we have our DNA samples,
[00:01:45.760]we're ready to go.
[00:01:46.630]So, we put everything in the tube.
[00:01:50.140]Our buffer that will do the chemical reaction,
[00:01:54.290]which is gonna be DNA replication,
[00:01:56.850]the DNA polymerizing enzyme,
[00:02:00.140]the nucleotides,
[00:02:01.630]and a little bit of mineral oil
[00:02:03.330]which will seal up the tube
[00:02:05.100]and make sure we don't lose volume
[00:02:09.554]as we heat up the tube.
[00:02:11.129]And you'll see where that comes in.
[00:02:12.770]Okay, so we're gonna zoom in on those components
[00:02:15.060]that are inside of our test tube.
[00:02:20.820]Now we're ready to think about the very first
[00:02:23.179]part of the DNA
[00:02:26.190]analysis process of PCR.
[00:02:28.868]The DNA is being shown here as
[00:02:31.600]a double stranded DNA molecule,
[00:02:33.510]and that's really important that you
[00:02:34.863]know a little bit about DNA structure.
[00:02:39.650]Because of these two strands that make up the DNA molecule
[00:02:45.350]the process of replication happens in a very specific way.
[00:02:50.060]It happens in our cells this way and,
[00:02:52.350]as it turns out, you can have it occur in a test tube.
[00:02:58.620]Entirely based on the structure of these DNA strands.
[00:03:05.390]Well, let's let the animation kind of go to work.
[00:03:08.490]So, the key is
[00:03:10.130]these test tubes that contain the double stranded DNA
[00:03:13.540]are heated up.
[00:03:15.290]And what the heating does is it
[00:03:18.162]adds enough heat to break up hydrogen bonds
[00:03:21.980]that hold the two DNA strands together.
[00:03:24.850]So, the two strands,
[00:03:27.144]which are made up of a specific sequence of
[00:03:30.570]A's, T's, G's and C's,
[00:03:32.460]the individual sub-units of DNA,
[00:03:36.170]will no longer be a double stranded.
[00:03:38.850]You can see here it's now two single strands.
[00:03:41.710]The heat broke the hydrogen bonds
[00:03:43.750]that held the two strands together.
[00:03:45.860]Alright, so after we've heated up our tube
[00:03:48.280]all those components are still there.
[00:03:51.190]The red sequence is here,
[00:03:53.520]that's our DNA primer,
[00:03:54.960]so we had to order that to be made by a company
[00:03:58.460]that could make up a specific sequence of nucleotides.
[00:04:02.380]That sequence is gonna match up to a part of the gene
[00:04:05.540]that we want to have copied from our DNA sample.
[00:04:08.970]Then we have the individual nucleotides,
[00:04:10.750]and there's that Taq polymerase enzyme.
[00:04:13.300]So, let's see how they all work together.
[00:04:15.878]The test tube is cooled down.
[00:04:18.560]Okay, so when it cools
[00:04:20.470]the primer, because we had lots of copies of the primer,
[00:04:24.100]the primer will tend to match up with
[00:04:26.020]the original DNA sample.
[00:04:27.670]You can see we've got a strand here
[00:04:30.470]and we've got a strand here
[00:04:32.610]that have been primed.
[00:04:34.280]That means the primer's bound,
[00:04:36.350]and that lets the next step of DNA replication occur.
[00:04:40.370]What happens next is the DNA polymerizing enzyme
[00:04:43.760]will bind to that free prime end,
[00:04:46.830]a specific end,
[00:04:48.480]and start to read the template.
[00:04:50.910]And what's it doing?
[00:04:51.743]Yeah, it's putting in the complimentary sequence.
[00:04:53.884]I'm gonna go back,
[00:04:55.295]that happened pretty fast.
[00:04:57.298]It'll read the template, read the original,
[00:05:00.220]and put in a compliment.
[00:05:02.039]Now, I'm gonna go one more time,
[00:05:03.770]see what's going on?
[00:05:05.350]Okay, so
[00:05:06.730]we started with just one double stranded DNA molecule.
[00:05:10.400]Now what do we have?
[00:05:12.210]Two double stranded DNA molecules.
[00:05:14.464]So, the polymerase chain reaction does the polymerizing,
[00:05:19.160]just like DNA's replicated in our own cells.
[00:05:22.540]But, then it's a chain reaction, okay.
[00:05:24.810]The double stranded DNA is then heated up in our test tube,
[00:05:29.260]because we have it in this machine that heats and cools.
[00:05:32.600]The Taq DNA polymerase that's in the tube
[00:05:37.817]can do the polymerization.
[00:05:39.450]Now, the polymerization happened
[00:05:41.460]because we added a lot of those primers.
[00:05:43.340]They bound again, the DNA polymerase took off.
[00:05:46.230]See that?
[00:05:47.380]Okay.
[00:05:48.240]And so, every round of heating and cooling
[00:05:51.707]doubles the amount of DNA in our test tube.
[00:05:55.120]So, it's very specific.
[00:05:56.690]We're not making copies of all the DNA,
[00:05:59.020]just the DNA from a particular segment
[00:06:01.980]of a gene that we're interested in.
[00:06:04.160]And, once we have enough copies made
[00:06:06.306]in this test tube,
[00:06:08.620]we can then do our next analysis called
[00:06:11.700]gel electrophoresis, and actually look at those copies.
[00:06:17.090]Okay.
[00:06:17.923]So, this is Don Lee,
[00:06:19.260]and I appreciate the chance to
[00:06:22.050]describe this polymerase chain reaction to you.

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Polymerase Chain Reaction (PCR) in Pigs

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