The Steps of PCR

in this video, we're going to talk about the steps of PCR, or polymerase chain reaction. And so PCR is actually a cyclical process that occurs in cycles where each of the cycles is going to have three steps that we have numbered down below. And so the very first step within each cycle is going to be the step of D natural ation, which is going to occur at high temperatures. The second step within each PCR cycle is going to be a kneeling, which is going to occur at low temperatures, and then the third and final step within each PCR cycle is going to be extension, which is going to occur at moderate temperatures. And so as we move forward in our course, we're going to talk about each of these three steps within each PCR cycle in more detail. But these three steps are going to be repeated in each PCR cycle, generating an exponentially growing number of DNA molecules and so down below. In this image, what we're showing you are the steps of PCR within just one cycle, and so at the very, very beginning, we're of course, going to have our reactant us and all of the four components that we talked about in our previous lesson videos, which includes the template D N A. The PCR primers, the heat resistant or thermo stable DNA preliminaries, tack, preliminaries and also the four nucleotides, the DNA nucleotides. Those would all be in part of the reactant within the test tube. And so, of course, it's going to include the template DNA, which is the gene of interest, which is what we're focusing on right here. And so in the very first step of the PCR cycle, we have DNA saturation, which is going to occur at higher temperatures. And so the D N A. Is going to be heated until the d n a d natures. And so the DNA maturation of the D. N A. Is going to separate the two strands of DNA, and so you can see that we have these flames here indicating the increase in temperature that is going to denature the D. N A. And separate the two strands. You can see that the hydrogen bonds have been broken and the two d N A strands have been separated, allowing each of those DNA strands to serve as a template. And so, in the second step of each PCR cycle, what we have is the process of a kneeling, and so this is going to occur at cooler temperatures. And so the DNA primers are going to bind with the single stranded DNA A at these cooler temperatures, and so you can see that now the DNA primers have bound to the DNA, and they are opposite Lee oriented and facing towards each other in terms of their five prime and three prime ends pointing towards each other. And so then we have the process of extension, which is going to occur at moderate temperatures. And so the heat resistant or the thermo stable DNA preliminaries called Tak preliminaries is going to build new D N A strands. And so it's going to be extending off of the primers and that five prime 23 prime direction for both strands and it will be building and amplifying the DNA. And so what we end up with are the products at the end of the first cycle of PCR, and so the gene of interest is going to have been amplified at the end of the PCR cycle and so you can see that now we have two copies. Two identical copies of the original gene of interest. And so again, this PCR, uh, steps. These PCR steps occur in cycles. And so these two here would be subject to the same process DNA creation and kneeling extension over and over and over again in these sets of cycles. And so again, we'll be able to talk about each of these steps within each cycle and more detail as we move forward in our course. But for now, this here concludes our brief introduction to the steps of PCR DNA saturation, a kneeling and extension, and I'll see you all in our next video.

in this video, we're going to focus on the first step of PCR, which is Dina maturation. And so, in the very first step of PCR cycle, heat is going to be used to denature the d N A. And so we're going to heat denature the double stranded DNA in order to convert the D N A into its single stranded form. And each of the single stranded DNA molecules can serve as a template for a new molecule DNA. And so the temperature of the PCR make sure is going to be increased, and it's going to be increased to a temperature of about 95 degrees Celsius, which is very near boiling temperatures and increasing the temperature to 95 degrees Celsius is going to help break all of the hydrogen bonds or the H bonds between complementary base pairs in the DNA and breaking those hydrogen bonds is going to separate the double stranded DNA into its single stranded form. Now it's very important that a special thermo stable DNA preliminaries, such as tack preliminaries, is used in the PCR mixture, and this tack preliminaries is not going to denature at these high temperatures, so it does not denature at high temperatures that are used in PCR, and so the attack preliminaries is able to withstand extremely high temperatures. But it's also able to withstand rapid temperature decreases as well, and we're going to see rapid temperature decreases in the second step of PCR. However, it's also really important to note that although tact preliminaries can withstand high temperatures and can also understand rapid temperature decreases, um, tactile memories does not actually synthesized d n A. Unless it is at an ideal temperature. And so this will be important once we get to the third step. And so, in the very first step of PCR, what we have is Dina maturation, and so within our test tube, we're going to have our d n A. And we'll need to raise the temperature which you can see. These flames here are going to raise the temperature and the heat is going to de nature. So the heat D nature's the complementary DNA strands and denature during the d. N. A really just means that the hydrogen bonds are going to be broken and the D N A is going to be forming its single stranded form. So you can see we have two single stranded DNA molecules here. And so again, within this test tube, we need to ensure that there is a thermo stable, uh, preliminaries. And so you can see over here what we have is our thermo stable preliminaries here, and you can see he's got this name tag that says hello. My name is Tech, because this is tak proliferates and notice. Attack preliminaries has no problems with the heat. The heat never bothered him anyway. And so you can see here that tactile memories is going to be very, very important to have and use during PCR. And so this here concludes our very, uh, our introduction to the first step of PCR DNA saturation. And as we move forward, we'll be able to talk about the 2nd and 3rd steps of PCR. So I'll see you all in our next video.

in this video, we're going to talk more about the second step of each PCR cycle, which is a kneeling. And so in this second step, the DNA primers are actually going to a kneel to the heat denatured single stranded DNA. And so, in this second step of PCR, a kneeling of the DNA primers to the heat denatured single strand of D N A. Is going to occur, and so the temperature is going to be lowered down to about degrees Celsius. And so, if you recall and step number one, the temperatures were as high as 95 degrees Celsius, and so there is a significant drop from 95 degrees Celsius down to about 55 degrees Celsius. And so that's lowering the temperature. And this lowering of the temperature allows for complementary base pairing. And so the complementary base pairing is going to occur between the DNA primers and the single stranded DNA itself. Now attack preliminaries, uh, is actually going to remain inactive at these cooler temperatures because the temperatures are just too cool or too cold for it to synthesize DNA. And so when we take a look at our image down below, Over here on the left hand side at the second step a kneeling. What you can see is that the temperatures are going to be cooled, so cooler temperatures are going to allow the DNA primers to a kneel to the d N A. And so, after the first step of PCR, which is going to be denatured aeration, we know that we have generated single stranded DNA, this heat, denatured DNA. And so in the second step, the temperatures are cold so that the DNA primers can a kneel to the d n A. As we see here and notice that they are kneeled on different DNA strands and they are kneeled, facing towards each other in terms of their five prime and three prime ends. This one goes from left to right, and this one goes from right to left facing towards each other. And so now that these DNA primers have been a kneeled, uh, and the third step, they can be extended. However, it cannot be extended here in the second step because these temperatures of degrees Celsius are just too cold for the tack preliminaries to work. And so notice the tactile memories is saying, Burr. I can't work like this and he's all cold. And he can only work when we increase those temperatures just a little bit to the right temperatures. And so this year concludes our brief introduction to the second step of PCR, a kneeling of the DNA primers, and we'll be able to talk about the third and final step of PCR in our next video.

in this video, we're going to talk about the third and final step of each PCR cycle, which is extension. And so the final step of PCR, which is this third step, is the extension of the new DNA strand by the thermo stable tack prelim a race. And so the temperature in the third step is going to be changed. An increase from 55 degrees Celsius in the second step to about 72 degrees Celsius here in the third step. And the 72 degrees Celsius is an ideal temperature for the activity of the tech preliminaries. And so the tact polyamorous is going to incorporate deoxyribonucleic tides and or those DNA nucleotides and use them to extend the DNA primers on each strand of the DNA making two identical copies of the D. N A. And so if we take a look at our image down below at this third step here of the PCR uh, cycle extension, which will notice, is that over here on the left, we're starting off with what we ended within the second step, which is the a kneeling of those DNA primers. And so here in the second step, the temperature is going to be increased to the ideal temperature for tact. Kolyma race. And so the attack preliminaries is able to incorporate the DNA nucleotides the free DNA nucleotides and use those free DNA nucleotides to extend each of the primers. And so you can see that the extension of the primaries is always going to be in the five prime 23 prime direction. And so the tactile memories will extend this and this direction. And the tactile memories here will extend this primer in this direction from right to left. And so the tack preliminaries extends the new strands of D N A. Until we have the template d n A. Which has been replicated. So the template d n A. Has been replicated here. And so you can see that the original we now have two identical copies of the original template D N A. And so that is going to conclude this third and final step of each PCR cycle. And so after, uh, the third step has been completed, then it can go back into a new cycle of PCR, where each of these products from the first cycle can be used as reacted in the second cycle and so on. And so this year concludes our introduction to the third step of PCR extension, and we'll be able to get some practice applying all of the concepts that we've talked about as we move forward in our course, So I'll see you all in our next video.