hello everyone. In this video, I'm going to speak about a really interesting technique used very widely nowadays, which is PCR (polymerase chain reaction). I'm going to speak about everything: the principle, the technique, the procedure, everything. So let's start by saying or by asking, what is PCR? PCR is a technique of DNA amplification. So what is the enabling patients? Let's say we have a DNA. We extract a DNA from a human cell, and we are interested in a particular gene or a particular DNA sequence. Let's say we want to examine it, we want to analyze it, we want to clone it, we want to do anything with this sequence. So if we have one copy of this sequence, it's, of course, it's not enough to do anything, but we want to copy this sequence. We want a large or a huge number of copies of this particular sequence. In this case, we use PCR because PCR can give us millions of copies out of a particular sequence of DNA. And in a short time, in like one hour, we can

get millions of copies of this particular gene or a particular sequence. Now, how did the technique work? If I want to say one word about PCR, PCR is a thermal cycle. What is a thermal cycle? So, I can say it's an increase and an edit, and increase and decrease in temperature. So, we heat up and we cool down our mixture in order to produce multiple copies of DNA. So, it's like this. So, let's say this is temperature. We increase or we heat up the temperature, then we cool down, then we heat up again, then we cool down, and in a cyclic way as you see here. So, this, let's say this is one cycle, this is another cycle, this is the third cycle, and so on. So, it's a thermal cycle. Of course, this is very, very, very general. I'm going to speak about everything in details in a few minutes. Okay, so generally or principally, PCR is a thermal cycle. It depends mainly on temperature. Okay, so let's say this is our tube. We want to perform PCR in this tube. What should we

have in this tube. So let's say PCR is DNA generates generation because it's DNA synthesis. We are copying. We're copying a particular sequence of DNA. First of all, of course, we should have the parent DNA. We should have our sequence of interest or the DNA sample. Okay. Now we need to copy this particular sequence. We want to synthesis new sequence of or new sequences of DNA. In this case, we need, of course, the DNA contents. In the DNA contents are the deoxynucleotide 3 phosphates, which are DNA contents. The cytosine guanine adenine timing and the deoxynucleotide triphosphate looks like this. So we have the deoxyribose sugar and we have the bases here: one base adenine, thymine, guanine, cytosine. We have three phosphates. So the generation of DNA will or the sentences of DNA will take place starting from these materials. Um, third, what should we have? So now I have the parent DNA. I have DNA content. In order to send us a new, the DNA, I need

something very important, which is the primer, in order to point on the particular sequence of DNA. I want to copy, so the primer is very specific to the sequence order to the gene I want to copy. Primer is, I can say, it's a starter of DNA synthesis or did a copying of the particular sequence for PCR. I need a couple of primers, the forward and the reverse draw primer for the two complementary sequences of DNA, and also you're going to see this in details in few minutes, but yeah, keep in mind I need two primers, forward and the reverse primer, and forth, of course, I need something for DNA synthesis, very interesting, very important thing, which is DNA polymerase, of course, DNA polymerase, and PCR should be heat-resistant because, as I told you, PCR is a thermal cycle, so it depends on heat, on on temperature, and I need to increase the temperature in PCR, so I need DNA polymerase to be his dentist Center resistant. There is one known PCR DNA polymerase, which is the tag polymerase.

but there are others, of course, but that polymerase is one very known polymerase for the PCR. Okay, so these are the four essential components I need to start a DNF a PCR. Now, what is the procedure? How that, how does PCR work or the steps? I told you PCR is a thermal cycle, so the thermos in it. So it's a thermal cycle, which is repeated, repeated several times. But in each thermal cycle, we have three steps of temperature changes. So in each term, in each one cycle, in each cycle, which are the temperature three times. So now I'm going to speak about the three steps of the thermal cycle or the of the PCR cycle, which are first, first step is the denaturation of hydrogen bonds, denaturation of the DNA. What does that mean saying we have this DNA, okay, this is these are double, double strand, this is double-stranded DNA, and um, we need to copy this particular sequence, okay, this specific sequence, of course. First of all, I should separate the two strands from each other, and as you know

there are hydrogen bonds here between the two by a binding the two strands together. So I need to break or two broke these hydrogen bonds. How does this does it work with heat with temperature so this is the denaturation of hydrogen bonds so this is let's say the particular sequence I need to to copy. So the two strands are bound together by hydrogen bonds and to break the hydrogen bonds I need I need heat so I increase the temperature. I heat up my PCR mixture to 94 between 94 and 98 degrees of Celsius for 20 to 30 seconds. Okay after 20 to 30 seconds the hydrogen bonds will be broken so I will have two separated strands of DNA um normally the the temperature here and the time depends on the GC content of the sequence why because as you see here the guanine and cytosine bind together with three hydrogen bonds yls diamond and adenine bind together with two hydrogen bonds so the GC bond is stronger than the TA bond and because of this is that when I have

higher GC content in my sequence. I need more time. I need like 30 seconds, and I need higher temperature in order to break the GC bonds. Okay, so this is the first step, the denaturation of hydrogen bonds. Now we have two separated strands. At the end of the first step, we have two separated strands of DNA, and then we go to the second step. In the second step, which is the annealing step. In the second step, so now we have two separated strands of DNA. Okay, so this is five prime three prime, this is three prime five prime, these two strands are complementary. In the annealing step, there is the hybridization of the primers with the DNA strands. So these are the primers, the primer normally is between 18 and 30 nucleotides, and the primers bind to the DNA or hybridize with the DNA exactly upward the sequence, the particular sequence in the three prime side of the sequence. And why, why the primer always is deposited on the three prime side of the sequence, because the synthesis

of DNA takes place always from five prime to three prime, so we need the copied strand or the copy the copied sequence sentences to take place from five prime to three prime. Okay, so this is the forward, this is the Reavers. I told you we have to price a couple of primer. The forward is for the first strand, the Reavers is for the complementary strand. So the synthesis of DNA should take place from five prime to three prime. If you want to know why the synthesis of the DNA takes place always from five prime to three prime, I explain this clearly in a previous video. You can find the link in the description of this video, so go to the other video and watch it. You can understand why the synthesis of DNA takes place always from five prime to three prime. But what you not you need to know at the moment is that DNA synthesis takes place always from five prime to three prime, and because of this, the primer hybridizes with the DNA on the three prime side of the sequence just upstream the

the sequence of it of interest now. For this step, I need to cool down the temperature to 50, between 50 and 65. If you remember, in the first step, the temperature was between 94 and 98. Now, the temperature is between 50 and 65 degrees Celsius for 20 to 40 seconds. The temperature here depends on the primer. Why? Because the primer needs a particular temperature in order to hybridize with the DNA. So, if the temperature is too low, the primer would not bind to the DNA. And we don't need the primer just to bind to the DNA. We need the primer to bind specifically to the upstream target. So, we need higher than 60% match between the primer and the DNA sequence in order to be specific. We don't need the primer to bind here or there. We need the primer to bind specifically upstream the target. Okay, so we need higher than 60% match. And because of this, we cannot raise the

temperature. If so, I told you, if we have cool temperature, the primer will not bind, and if we have a high temperature, the primer will bind. None specifically. The primer would bind here, for example. None specifically. And we don't need this. So there is a rule of thumb we can say. Normally, for this step, we use a temperature which is 4 to 5 degrees lower than the melting temperature of the primer. So let's say we have a primer whose melting temperature is 60. In this case, we use 55. Okay. Now, this is the annealing step. In the annealing step, it's nothing but the hybridization of the primer with the DNA sequence or with the DNA strand. Now, going to the third step. In the third step, the binding of DNA starts. So it's the elongation step. Here we have nucleotides and DNA, and the copying of DNA starts in this step. So here we have the primer. So let's say this is the sequence of the primer. I told you the primer is something between 18 and 30 nucleotides.

the primer provides a hydroxyl group. This is the hydroxyl group needed to build up the new nucleotides. The hydroxyl group is on the three prime end of the primer. Yeah, again, you can find all these details in the previous video, whose link is in the description of this video. So, the hydroxyl group is needed to start the DNA synthesis from five prime to three prime. And of course, in this step, we need something very important, which is the DNA polymerase. And we need a particular, of course, a particular temperature, because PCR is all about temperature. So, we need a temperature between 75 and 80 degrees Celsius, where we apply it for around 2 minutes, and then the polymerase will start to build the new copy of DNA. Okay, at the end of the elongation step, we will get two separated copies of the DNA of the particular sequence of DNA we need. For the two minutes are variable, they depend on the length of the sequence.

normally, we need 1 minute for each 10-bay tent out for each thousand bases, but yeah, it's somehow around 2 minutes. So, at the end of this step, as I told you, we will get, we will have two separated copies of the particular sequence of DNA. If it is so, these are the three steps, and if we want to look at the thermal cycle all together, it looks like this. So, this is the PCR thermal cycle. So, it starts by heating up the temperature, starting the DNA denaturation, the DNA denaturation steps, step. Then we cool down the temperature in order to, as I told you, so in the DNA denaturation, as I told you before, we separate the two strands of DNA from each other, and it's around, as I told you, it's around 94 to 98 degrees Celsius. Then we cool down to the temperature for the primer annealing. We need around 50 to 65 degrees Celsius. In this step, the primers hybridize with the DNA, the forward and the reverse.

primer hybridize to the DNA on the 3 prime 3 prime side of the of the sequence. Then we heat up the temperature again for DNA extension, in which the new copy of DNA is tested synthesized, and the elongation of DNA takes place from 5 prime to 3 prime end. At the end of the DNA extension, at the end of the first thermal cycle. So thermal cycle is three steps. This is the end of the thermal cycle. Then we heat again. We have denaturation again. Primer here, here at the end of the second thermal cycle. Ok, we can have as much thermal cycles as we wish. So what I want to tell you that at the end of the first thermal cycle, we get two copies of the needed sequence. And then when we start with the second thermal cycle, each one of these two will be copied to the to another two. So, at the end of the second thermal cycle, we get two copies from each copy. So we get four copies at the end of the of the second thermal.

cycle. So let's see how does it work. So start with one copy with one sequence. At the end of the first cycle, we have two sequences. At the end of this second cycle, we have four. And then from four, we have eight. And then from eight, we have sixteen. And so on. So it's like a cascade. Now, there is an so how many copies I will have at the end. I will the number of copies. I I will I would get at the end is 2 to the power n. N in the number of cycles. So let's have a I can give you an example. Okay. So let's say we run the PCR for one hour. Okay. What would we get if we run the PCR for one hour. So let's say we run the PCR for one hour. So if one thermal cycle takes around two to three minutes. Okay. Let's say it takes around two to three minutes. Three minutes. So in one hour, I will have 20 cycles. And then the number of copies will be 2 to the power 20, which is 1.

million for you. 1,048,576. So, imagine after one hour of PCR, I will get over 1 million copies out of one copy. After a out of one copy of DNA, which is cool, which is pretty cool. With one with this 1 million for 48 thousand, I can I can do whatever I want to do. I can clone the sequence. I can study it. I can analyze it. So, let's say what applications of PCR do we have. We can do DNA isolation. We can isolate a particular gene. We can do DNA quantification. We can verify the presence of the gene. We can do gene testing in order to see if we have mutation gene matching the oncogenes. We can do for viral and bacterial infection diagnosed like HIV, for example, or tuberculosis. We can use PCR for forensics, of course, because we can amplify the DNA we have. We can amplify it. So, there are many, many. These are a sample of applications of PCR, but in reality, we have hundreds of application of PCR. PC

um this is everything I wanted to tell you about PCR. I hope you understand this video. And there will be another video about real-time PCR. So if you liked the video, like. If you benefit from this video, like the video. Share it with your friends. Subscribe to my channel because there will be always new videos. And thank you for watching. See you in the next video. Bye.

The principle of PCR-Polymerase Chain Reaction, a full and easy explanation

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The principle of PCR-Polymerase Chain Reaction, a full and easy explanation

The principle of PCR-Polymerase Chain Reaction, a full and easy explanation

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