Polymerase Chain Reaction (PCR) in Pigs

Don Lee, Presenter Author

12/18/2018 Added

360 Plays

Description

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.

Searchable Transcript

Search:

[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.

The screen size you are trying to search captions on is too small!

You can always jump over to MediaHub and check it out there.

Comments

0 Comments

Polymerase Chain Reaction (PCR) in Pigs

Description

Searchable Transcript

Comments icon comment

Related Channels

Comments