Polymerase Chain Reaction (PCR) in Plants

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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[00:00:08.190]Polymerase Chain Reaction,
[00:00:09.960]or PCR as it's often called,
[00:00:12.750]is an incredibly powerful DNA analysis technique
[00:00:16.670]that is based on the process of DNA replication.
[00:00:22.600]So, I'm gonna give a little narration of this
[00:00:25.760]animation developed by a previous USDA project
[00:00:30.760]at University of Nebraska,
[00:00:32.700]and let's see if we can understand how PCR works.
[00:00:35.570]So, we're gonna start with getting at the DNA
[00:00:39.053]that is part of chromosomes that are inside the plant cell,
[00:00:45.506]that make up tissues that we sample.
[00:00:48.210]So, if we sample a little leaf
[00:00:50.230]we're gonna get the chromosomes
[00:00:52.960]that contain DNA from the leaf.
[00:00:55.160]But, it really doesn't matter
[00:00:56.580]what part of the plant we sample,
[00:00:58.070]the same DNA is found in all parts of a plant.
[00:01:02.500]So, the key
[00:01:05.060]with PCR is that
[00:01:06.680]we're gonna make copies of just a specific segment of DNA
[00:01:11.730]from all the DNA that we extracted out of our tissue.
[00:01:16.890]You're gonna see how we can be very specific about that.
[00:01:20.570]Now, before we can do PCR
[00:01:23.370]we have to obtain our DNA sample,
[00:01:25.900]and then we have to do a little shopping.
[00:01:27.770]We have to buy an enzyme called Taq polymerase,
[00:01:31.870]we have to buy the individual DNA nucleotides
[00:01:34.940]that will be polymerized into new copies of DNA.
[00:01:40.540]We need to buy what's called a DNA primer,
[00:01:43.080]you'll see where that will fit in,
[00:01:44.520]and we have to buy some test tubes and reaction buffers.
[00:01:49.650]Okay.
[00:01:51.100]Our lab is well stocked,
[00:01:52.530]we have our DNA samples,
[00:01:53.710]we're ready to go.
[00:01:54.580]So, we put everything in the tube.
[00:01:58.090]Our buffer that will do the chemical reaction,
[00:02:02.240]which is gonna be DNA replication,
[00:02:04.800]the DNA polymerizing enzyme,
[00:02:08.090]the nucleotides,
[00:02:09.580]and a little bit of mineral oil
[00:02:11.280]which will seal up the tube
[00:02:13.050]and make sure we don't lose volume
[00:02:17.504]as we heat up the tube.
[00:02:19.079]And you'll see where that comes in.
[00:02:20.720]Okay, so we're gonna zoom in on those components
[00:02:23.010]that are inside of our test tube.
[00:02:28.770]Now we're ready to think about the very first
[00:02:31.129]part of the DNA
[00:02:34.140]analysis process of PCR.
[00:02:36.818]The DNA is being shown here as
[00:02:39.550]a double stranded DNA molecule,
[00:02:41.460]and that's really important that you
[00:02:42.813]know a little bit about DNA structure.
[00:02:47.600]Because of these two strands that make up the DNA molecule
[00:02:53.300]the process of replication happens in a very specific way.
[00:02:58.010]It happens in our cells this way and,
[00:03:00.300]as it turns out, you can have it occur in a test tube.
[00:03:06.570]Entirely based on the structure of these DNA strands.
[00:03:13.340]Well, let's let the animation kind of go to work.
[00:03:16.440]So, the key is
[00:03:18.080]these test tubes that contain the double stranded DNA
[00:03:21.490]are heated up.
[00:03:23.240]And what the heating does is it
[00:03:26.112]adds enough heat to break up hydrogen bonds
[00:03:29.930]that hold the two DNA strands together.
[00:03:32.800]So, the two strands,
[00:03:35.094]which are made up of a specific sequence of
[00:03:38.520]A's, T's, G's and C's,
[00:03:40.410]the individual sub-units of DNA,
[00:03:44.120]will no longer be a double stranded.
[00:03:46.800]You can see here it's now two single strands.
[00:03:49.660]The heat broke the hydrogen bonds
[00:03:51.700]that held the two strands together.
[00:03:53.810]Alright, so after we've heated up our tube
[00:03:56.230]all those components are still there.
[00:03:59.140]The red sequence is here,
[00:04:01.470]that's our DNA primer,
[00:04:02.910]so we had to order that to be made by a company
[00:04:06.410]that could make up a specific sequence of nucleotides.
[00:04:10.330]That sequence is gonna match up to a part of the gene
[00:04:13.490]that we want to have copied from our DNA sample.
[00:04:16.920]Then we have the individual nucleotides,
[00:04:18.700]and there's that Taq polymerase enzyme.
[00:04:21.250]So, let's see how they all work together.
[00:04:23.828]The test tube is cooled down.
[00:04:26.510]Okay, so when it cools
[00:04:28.420]the primer, because we had lots of copies of the primer,
[00:04:32.050]the primer will tend to match up with
[00:04:33.970]the original DNA sample.
[00:04:35.620]You can see we've got a strand here
[00:04:38.420]and we've got a strand here
[00:04:40.560]that have been primed.
[00:04:42.230]That means the primer's bound,
[00:04:44.300]and that lets the next step of DNA replication occur.
[00:04:48.320]What happens next is the DNA polymerizing enzyme
[00:04:51.710]will bind to that free prime end,
[00:04:54.780]a specific end,
[00:04:56.430]and start to read the template.
[00:04:58.860]And what's it doing?
[00:04:59.693]Yeah, it's putting in the complimentary sequence.
[00:05:01.834]I'm gonna go back,
[00:05:03.245]that happened pretty fast.
[00:05:05.248]It'll read the template, read the original,
[00:05:08.170]and put in a compliment.
[00:05:09.989]Now, I'm gonna go one more time,
[00:05:11.720]see what's going on?
[00:05:13.300]Okay, so
[00:05:14.680]we started with just one double stranded DNA molecule.
[00:05:18.350]Now what do we have?
[00:05:20.160]Two double stranded DNA molecules.
[00:05:22.414]So, the polymerase chain reaction does the polymerizing,
[00:05:27.110]just like DNA's replicated in our own cells.
[00:05:30.490]But, then it's a chain reaction, okay.
[00:05:32.760]The double stranded DNA is then heated up in our test tube,
[00:05:37.210]because we have it in this machine that heats and cools.
[00:05:40.550]The Taq DNA polymerase that's in the tube
[00:05:45.767]can do the polymerization.
[00:05:47.400]Now, the polymerization happened
[00:05:49.410]because we added a lot of those primers.
[00:05:51.290]They bound again, the DNA polymerase took off.
[00:05:54.180]See that?
[00:05:55.330]Okay.
[00:05:56.190]And so, every round of heating and cooling
[00:05:59.657]doubles the amount of DNA in our test tube.
[00:06:03.070]So, it's very specific.
[00:06:04.640]We're not making copies of all the DNA,
[00:06:06.970]just the DNA from a particular segment
[00:06:09.930]of a gene that we're interested in.
[00:06:12.110]And, once we have enough copies made
[00:06:14.256]in this test tube,
[00:06:16.570]we can then do our next analysis called
[00:06:19.650]gel electrophoresis, and actually look at those copies.
[00:06:25.040]Okay.
[00:06:25.873]So, this is Don Lee,
[00:06:27.210]and I appreciate the chance to
[00:06:30.000]describe this polymerase chain reaction to you.

Polymerase Chain Reaction (PCR) in Plants

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