RT-PCR: The Taqman System

Don Lee Author

05/27/2016 Added

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For more information please follow the link: https://ge.unl.edu/oomycete-disease-diagnostics/

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[00:00:05.905]Hello, this is Don Lee
[00:00:07.007]from the University of Nebraska at Lincoln.
[00:00:09.782]And I will narrate an animation
[00:00:13.997]that illustrates the process of Taqman or quantitative PCR.
[00:00:18.983]I'll transition here to the animation.
[00:00:24.730]It was developed with a previous grant
[00:00:27.770]as supported by the USDA.
[00:00:30.453]And you'll see that this particular version
[00:00:33.577]of Taqman PCR
[00:00:37.129]shows its potential application to detect a transgene
[00:00:41.801]that might be present in a grain sample.
[00:00:45.112]But the same process would occur
[00:00:49.345]in any particular DNA sample
[00:00:52.656]a scientist might be interested in working with
[00:00:55.968]where there are a number of different possible sequences
[00:00:59.692]that can be detected and they need to be able
[00:01:04.058]to know something about the relative abundance
[00:01:06.903]of a particular DNA sequence.
[00:01:09.546]Okay, so
[00:01:11.762]the Taqman PCR is named
[00:01:14.766]after the machine that's used in the process,
[00:01:19.559]and the enzyme that's used
[00:01:22.110]to do the rounds of DNA replication.
[00:01:26.342]And you'll see that the
[00:01:29.127]PCR reaction is the same as the one
[00:01:32.351]that you've previously learned about
[00:01:34.567]that we might call conventional PCR.
[00:01:36.863]In conventional PCR you're making copies
[00:01:39.306]of a specific fragment of DNA,
[00:01:42.684]and then copies of the copies of the copies.
[00:01:45.448]And once you have enough copies
[00:01:47.397]you can visualize those by running the DNA
[00:01:50.615]through an electrophoresis gel,
[00:01:53.340]staining the gel,
[00:01:54.675]and then the presence of that DNA
[00:01:56.315]can be seen as a band compared to
[00:02:01.029]a sample that lacks the copies
[00:02:05.888]of the particular fragment that you're targeting.
[00:02:09.560]And so you get this yes no, plus minus answer
[00:02:13.446]with conventional PCR.
[00:02:15.035]Is a particular DNA sequence present, or is it absent?
[00:02:20.494]The difference with quantitative PCR is
[00:02:23.245]that you get an output that tells you
[00:02:27.211]if a particular fragment of DNA is getting copied,
[00:02:32.766]and you can get an idea of the relative amount
[00:02:37.118]of that DNA in your DNA sample
[00:02:41.069]by how quickly that amplification peak starts
[00:02:44.221]to be read in the output of this device.
[00:02:48.142]So we'll wanna understand how this output works
[00:02:51.244]by watching this animation.
[00:02:55.195]So, the procedure always starts
[00:02:57.906]by obtaining a sample of DNA.
[00:03:01.806]Can be from any source.
[00:03:05.850]And then you have to put all the components
[00:03:08.427]in a test tube that can allow for
[00:03:12.446]in vitro DNA replication.
[00:03:17.693]So you have to add in the...
[00:03:24.863]individual nucleotides that can be used
[00:03:26.798]to build DNA molecule.
[00:03:28.241]You have to add in two primers.
[00:03:30.444]The primers will bind to a specific DNA sequence,
[00:03:35.132]and if you select the right primers,
[00:03:37.067]you can initiate the copying of the DNA
[00:03:41.005]between where those two primers are binding.
[00:03:44.984]You have to have a DNA-polymerizing enzyme
[00:03:48.027]and usually this thermus aquaticus
[00:03:50.925]or TAQ polymerase is used
[00:03:53.328]because of its heat stability.
[00:03:55.372]And then in quantitative PCR you
[00:03:57.000]add in one more component, a probe
[00:04:00.085]that has a DNA sequence that can be incorporated into
[00:04:04.798]the copies of the specific DNA sequence
[00:04:08.683]that you're interested in.
[00:04:10.806]And this probe has a light-emitting biomolecule
[00:04:16.317]that you can use for detection.
[00:04:25.600]Alright, so let's see how all these components
[00:04:29.525]fit together in the quantitative PCR process.
[00:04:40.073]Alright the scientist will put all the components
[00:04:42.730]together in the same test tube
[00:04:44.438]or maybe they have a robot programmed
[00:04:46.735]to do all this pipetting for them.
[00:04:53.103]And then in PCR,
[00:04:56.441]you can have a setup that could involve a...
[00:05:02.224]plate that has 96 different compartments,
[00:05:06.363]so you could sample many samples all at the same time.
[00:05:13.251]And then within each of the test tubes
[00:05:15.294]you have what it takes
[00:05:17.110]for specific DNA replication to occur.
[00:05:21.409]And if the rounds of replication are occurring
[00:05:23.545]you can amplify a particular sequence.
[00:05:26.055]So just as in regular PCR
[00:05:28.031]you have your double-stranded DNA.
[00:05:30.702]And instead of adding an enzyme
[00:05:32.931]that would unwind that double-stranded DNA,
[00:05:36.803]these microtiter plates
[00:05:39.781]or racks of test tubes are placed
[00:05:42.384]into a special instrument, a thermal cycler,
[00:05:45.536]and that thermal cycler
[00:05:47.981]will heat up and cool down.
[00:05:50.582]And when it heats up,
[00:05:52.892]the double-stranded DNA is denatured.
[00:05:56.082]Here we're showing the two strands
[00:05:57.724]with their complementary sequences.
[00:06:00.155]And to make things as simple as possible,
[00:06:03.518]we're showing a very small segment of DNA.
[00:06:06.390]But you could have your primers bind
[00:06:08.004]to larger fragments of DNA,
[00:06:11.543]or smaller fragments of DNA.
[00:06:13.319]These primers are designed to target
[00:06:15.549]the sequence that you're interested in.
[00:06:18.553]So the heating up denatures the double-stranded DNA
[00:06:22.559]and then the instrument cools down,
[00:06:24.374]that allows the probes to bind.
[00:06:26.843]And they will bind in way where they're
[00:06:29.046]they find and then complement the particular sequence.
[00:06:35.897]A scientist may have to work
[00:06:37.258]in order to find primers that bind
[00:06:40.933]with the specificity and reliability
[00:06:43.519]that they're interested in.
[00:06:45.268]And then the third matching DNA sequence
[00:06:49.128]is that probe DNA sequence.
[00:06:51.984]You can see how the probe is designed to bind right here
[00:06:55.469]and that five to three prime orientation is important.
[00:06:59.475]So what happens then is
[00:07:01.384]that once the primers have bound,
[00:07:03.186]and the Taq DNA polymerase
[00:07:08.768]starts to do its work,
[00:07:10.351]on the strand where the probe binds,
[00:07:13.468]the Taq polymerase encounters the probe
[00:07:16.338]that is binding there on the same strand
[00:07:18.755]of DNA that the primer bound,
[00:07:20.730]and it will cleave off the first nucleotide.
[00:07:23.681]And that will emit a light-generating reaction.
[00:07:28.087]And then it will go ahead and continue
[00:07:31.384]to copy through where the probe was
[00:07:33.588]and finish the round of replication.
[00:07:37.820]And we can start with one copy
[00:07:40.009]of the double-stranded DNA and get two,
[00:07:42.561]and then as the cycles continue
[00:07:44.470]as long as we've added enough primers and probe
[00:07:47.501]all three will be involved in each round of replication.
[00:07:51.186]And as the copies get made, light is emitted.
[00:07:55.618]So, we can get a signal from this test tube
[00:07:59.183]in real time that goes along
[00:08:02.681]with the rate at which copies of DNA are being made.
[00:08:07.101]The more copies that get made,
[00:08:08.822]the more probes that get incorporated,
[00:08:10.959]the more light that gets emitted.
[00:08:15.311]Alright, so, remember these test tubes
[00:08:18.422]are being taken through rounds of replication
[00:08:21.935]inside our thermal cycler instrument.
[00:08:25.244]And if this is a Taqman PCR quantitative PCR instrument,
[00:08:30.372]it not only has the heating and cooling capabilities,
[00:08:35.765]it also has those, a light-detection capability.
[00:08:40.506]And so every single sample in your PCR run
[00:08:46.273]can be monitored for light emission.
[00:08:48.690]So in the early stages of the heating and cooling,
[00:08:52.468]we get just a very, very small amount of light emitted.
[00:08:57.238]So it's kind of not much over the background reading.
[00:09:02.962]But then once the copies start to get made,
[00:09:07.177]and then the copies of the copies
[00:09:09.184]of the copies are getting made,
[00:09:10.587]you get a stronger and stronger light emission signal.
[00:09:14.311]Until you reach a point where the primers
[00:09:17.769]and the probes start to run out
[00:09:20.480]and you lose the
[00:09:25.473]linear capability to see the increase
[00:09:28.490]in DNA copies being made.
[00:09:31.134]So, the more primer and probe you put in,
[00:09:33.777]the more light that you could get emitted,
[00:09:38.119]the higher this...
[00:09:41.921]band, this line will go.
[00:09:44.299]But the key to this analysis often is
[00:09:47.664]to compare the relative amount of DNA
[00:09:50.921]in one sample compared to a second sample
[00:09:53.965]compared to a third sample.
[00:09:56.008]Or you could have
[00:09:58.558]within the same test tube a number of different
[00:10:01.736]primers targeting different sequences occur.
[00:10:06.155]And you can see the difference between
[00:10:09.145]the amplification rate of a higher
[00:10:13.378]sequences present in higher copy number,
[00:10:16.061]compared to a little bit lower copy numbers,
[00:10:18.599]compared to lower copy numbers.
[00:10:20.120]So you can do a quantitative analysis
[00:10:24.300]to determine what the relative amount
[00:10:27.383]of a particular DNA sequence are in a DNA sample.
[00:10:32.604]So it's both a yes, it gives you both a yes no answer,
[00:10:35.635]yes I do see that that particular fragment present
[00:10:40.309]and being amplified in my test tube
[00:10:42.657]and a quantitative answer,
[00:10:45.182]there's more of it, or there's less of it.
[00:10:49.053]So that's how the PCR reaction works.
[00:10:54.620]You can set up experiments
[00:10:57.010]where you put in various amounts
[00:10:59.815]of particular DNA sequence and confirm
[00:11:03.927]that your instrument is quantitative in its output.
[00:11:07.927]And then you can apply this technique to unknown samples
[00:11:13.059]and the output tells you the relative amounts
[00:11:16.490]of a particular DNA sequence that might be present.
[00:11:21.201]So that's how quantitative PCR works.

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RT-PCR: The Taqman System

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