Chain-Termination PCR

by Jason Amores Sumpter
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in this video, we're going to continue to talk about di di Oxy sequencing as we talk about the chain termination PCR steps or the chain termination proliferates, chain reaction steps. And so recall from our previous lesson videos that we already talked about preliminaries, chain reaction or PCR. And so be sure to go check out those older videos on PCR polymerase chain reaction before you continue here. Now. Also, recall from our previous lesson videos that DNA synthesis reaction is actually terminated when a di di oxy nucleotide or a D d NTP is added to the three prime men of the growing DNA strand. And really the use of these d d N t. P s to terminate the chain is really what chain termination PCR relies on. And so, in the first two steps of deity oxy sequencing, it requires setting up a chain termination p c R, which is really just a PCR reaction that's going to include small amounts of D. D N T. P s. And so what you can see here is that in this first step, we're going to need to set up four separate reactions in four separate test tubes And so notice down below in our image over here on the left hand side, you can see that we've got these four different test tubes where we're going to set up four different reactions and each of these four separate reactions that are being set up, they're each going to contain all of the components that are needed for a normal PCR. And they're also going to contain a small amount of a different dd NTP and really, this small amount of different DD NTP is what distinguishes one tube from another test tube. And so what you can see here is that over here in this, uh, test tube, it has all of the components for a normal PCR. But it also includes the d d NTP for cytosine. And so basically, this is going to provide chain termination at all of the side. I've seen, uh, nucleotides upon, uh, amplification of the d N A. Then, in this test tube noticed that it also has all of the normal components, or PCR, but it differs from the previous one and that it has the d d NTP for third timing and so chain termination in this tube is going to occur at all of the timing nucleotides. Then what we have over here in this test tube is again. It's going to contain all of the components for a normal PCR. But it's also going to contain a small amount of the D d N T p for a Dinneen A's. And so the same goes for this one over here, this last test tube. It's going to have all of the components for a normal PCR, but it differs in that it has a small amount of the DDM TPS for wanting. And so these four test tubes differ from each other in the small amount of the different DD NTP. That's being added. And again, the D D NTP is going to lead to chain termination at that specific nucleotide season. The first two teas in the second A's in the third and GS in the fourth. And so what you can see up at the top here is the mystery, D n A. And the mystery D N A. Is the specific DNA sequence that we try, uh, that we want to be able to sequence and determine the sequence of this mysterious D n a whose sequence we do not know at the moment. And so die. The Oxy sequencing can help us determine the sequence of this mystery D n A. And we have to set up chain termination PCR. And so this mystery d n A. Is going to serve as the template DNA for amplification during this PCR. And so the mystery DNA is going to go into all four of these test tubes. Um, Then in step number two, what we have is conducting the actual PCR reaction the chain termination PCR reaction. And so DNA synthesis, um, is actually going to produce a bunch of fragments of D N A. And the reason that it produces fragments of D N A is because of the d d N T P s, which is going to terminate the DNA synthesis reaction and create a bunch of fragments that terminate at the specific nucleotides that are indicated in each to. And so the DNA synthesis produces fragments of DNA, and these fragments of DNA are going to be complimentary to the unknown target. And again, the unknown target is the mystery DNA that we have here and so down below in our image. What you can see is that upon step number two conducting the actual chain termination PCR, the D N A is going to be replicated with chain termination P c R. And so what you see over here are the products. The PCR products and which will notice is that there are a bunch of different sized fragments of DNA that are going to be generated. Some of them only have one nucleotide, others are going to have more nucleotides. And what you can see is that we have all of these different sized fragments and the one the frag. For each of these fragments, you'll notice that in the background there is like a different colored background at the end of each of these chains and the one that has that different colored background here, Uh, that would represent the D. V M T. P. That's being, uh incorporated and terminating the chain at that nucleotide. And so what you can see here is you get all of these different sized PCR products and again these PCR products are going to be complementary to the mystery d n A. And so you can arrange these PCR products so that they are arranged based off of the size of the fragment. And so the size of the fragment is actually going to be revealing the nucleotides of the complementary PCR products from five prime 23 prime end. And so if we focus mainly on these di di oxy nucleotides that are at the end that terminate the chain, the PCR, um, these nucleotides here are going to be the ones that allow for generating these different sized fragments. And so, um, we can actually analyze these PCR products and analyzing these PCR products, um, is something that we're going to talk about in our next lesson video. And when these PCR products are analyzed, it can actually reveal the sequence of the mystery DNA. And so this year concludes our introduction to the chain termination PCR, and we'll be able to get some practice applying this and then talk about exactly how this, um, these PCR products can be analyzed to reveal the sequence of the DNA in our next video. So I'll see you all there