Understanding Polymerase Chain Reaction Techniques

Hello and welcome back friends. In this video we will be talking about PCR or polymerase chain reaction. It is actually better known as PCR. Let's begin. As the name suggests, polymerase chain reaction, it means, let me discuss it to you. Polymerase means for this process we must have needed the polymerase enzyme, remember? the effect of polymerase enzyme during the DNA replication process it can add nucleotide sequences one by another and can elongate and can produce a nucleic acid chain like DNA or RNA. Now the polymerase can be of DNA polymerase and RNA polymerase both the case it can polymerize nucleotide sequences chain reaction that means the reaction procedures or sequential events in the reaction which will go on and on and on like a chain. So we are involving a polymerase enzyme for adding nucleotide sequences and making a long chain nucleotide sequences and the process will be a chain reaction process. That means we will amplify something. We are amplifying something with this chain reaction. Whenever we are talking about a kind of chain reaction, it means we are amplifying something. Now in this case also we are amplifying. The basic goal of utilizing polymerase chain reaction is to produce a copy of the segment of DNA in much more higher amount in high concentration. For example, say in this whole DNA content, this red colored segment of this gene is of our interest. This is the wanted gene. Now we want to amplify the number of this gene for our purpose, for our experimental purpose. So utilizing this polymerase chain reaction is allowing us to increase the number of that particular region of this DNA not the other regions of the DNA. And we can see after many cycles so it's a repeated process as you see in the name chain reaction so it is increasing the number of this template DNA of our interest in chain reaction or in higher magnitude in greater number as we go on in logarithmic spell and what we can get from one template of the DNA 2 then 2 to 4 then 4 to 8 and then 8 to 16 and so on it will start to generate a lot of number of this particular template DNA of our interest after many different cycles. This is the importance of PCR for the amplification of a particular segment inside the DNA. Suppose a desired region of our interest which is a gene a particular gene. It's an exponential amplification process. Now for this PCR reaction there are several steps of this pci reaction actually we can divide into three major steps of this pci reaction now it's all about the simple or basic mechanism of dna maturation dna naturation and denaturation okay dna naturation and denaturation is playing the important role here and also simple polymerization process that means the the dna polymerization process is also important so if you understand the dna polymerization process and denaturation of the dna you can understand this process pretty easily For example, say this is our desired DNA. Somewhere in between this desired DNA, we need to copy the gene. Right? So, suppose from this part to this part, we need to copy this gene. So, what we'll do, we will amplify this segment of this gene, but not amplifying the rest of the part. So, what we'll do in this case, the first step is the denaturation process. To denature the complementary strands of a DNA, we must heat it. So, we'll do that. at 94 degree Celsius temperature for about one minute. Now heating it in 94 degree Celsius temperature for one minute actually 90 or above 90 degree Celsius temperature for one minute it will denature the DNA and we are having this DNA in our hand. Now this is a host DNA and one thing you need to make sure before the denaturation or setting this denaturation temperature is that what is the Tm or melting temperature of the DNA. We know the melting temperature of the DNA varies from one species to another species. Right. Because the constituents of this nucleic acid can be varied. So the number of GC is very very important. GC content is very very important. We know this guanosine added with cytosine via the C hydrogen bonds. So they are much more stronger bonds. So we need to give much more energy to break GC bonds than to break the AT bonds. So that's why. we need to use high amount of temperature to break those DNAs or to separate those DNA strands which are having more GC than 80, right? So that's why we need to be very careful about the construction of the DNA. We need to look for the nucleotide makeup of the DNA before setting this temperature. Now let's say we set the temperature in this case 94 degree Celsius then what it will do? It will denature the DNA in a hole. After denaturing the DNA two strands are separated. So this is strand 1, this is strand 2. After this strand separation what we'll do is start providing some important oligonucleotide sequences. Stretch of oligonucleotide sequences. Why? Why we require this stretch of oligonucleotide sequences? Because remember what we need to do, we need to do a polymerase reaction, a polymerization reaction. And we know that DNA polymerase is only function, it can only function when we provide it a 3 prime hydroxyl. These are the basics of DNA replication process. I know, I hope you understand these things. If you don't just go back and look for the video of DNA replication and DNA polymerization videos in my website you can find it. Now these DNA polymers must have the C-Pyme hydroxyl to continue the polymerization events to provide it during the DNA replication also we need to attach some small sequence of primer. Now in this case also need to provide some oligonucleotide stretch which is called primer. Now we need to provide this primer and this primer will attach to this DNA segments flanking the region of our interest. Suppose this is the region of our interest. So, if we attach the primer flanked both the ways of our desired region of our interest. Okay, we give this primer. But one thing you need to make sure is that the primer sequences that we provide must have at least greater than 60% sequence similarities sequence complementarity, not similarity sequence complementary with the host DNS trans. otherwise it will not be able to bind with the host strand, right? So, we need to make sure this thing, okay? And also for the binding of the primer or for the annealing of the primer with the host DNA, we must lower down the temperature from 94 degree Celsius temperature to 45, sorry, to 54 degree Celsius temperature. And we allow this annealing process to happen in 45 minutes to, sorry, 45 seconds to 50 seconds like that, okay? So we require two different primers. One primer is called the forward primer. Another primer is called the reverse primer. Now in this case we attach, why is called forward and reverse? We know the polymerization step carries out from the 5'end to the 3'end. So those primers which are designed and attached to the 5'end to 3'end direction will be called the forward primers. For example, let me change the color here. Sorry, yeah. This one is a forward primer because it is testing to 5 prime end to 3 prime end and those primers which are getting from 3 prime end to 5 prime end the opposite direction it will be called the reverse primer So we get both the primers in this account We are getting forward primers as well as reverse primers Altogether we need to lower the temperature to the 50 to 54 degree Celsius temperature We need to allow it to happen for 45 seconds and then this primer will anneal with the host DNA sequence. Now after the anneal, remember the two strands are separated and we add primers both the strands. So utilizing both the DNA strands for amplification of our desired gene. Now after that we must add this all this nucleotide sequences there A, G, T and C because we require this nucleotide sequences to be attached with each other by the DNA polymerase. So nucleotide sequences will be attached using DNA polymerase. and we need to again rise the temperature a little bit to 72 degree Celsius temperature and this DNTPs will keep on binding and we need to carry out this for 2 minutes Okay, so this is the total process First denaturation in 94 degree Celsius temperature for 1 minute Second is annealing of primers at 54 degree Celsius temperature for 45 seconds to bind the forward and reverse primers After the binding of primers, we will provide the polymerase as well as the dNTPs to attach them and to polymerize these nucleotide sequences at 72 degrees Celsius temperature for 2 minutes and it will extend the primers and it will produce the segment of our interest or the gene of our interest. And this is called a one cycle of a polymerase chain reaction. So a denaturation, annealing and a renaturation and an extension, sorry, this whole procedure. is called the one cycle of this PCR or polymerase chain reaction. Now we keep on repeating these cycles to keep on repeating and producing our desired gene segments and amplifying our desired gene segments almost like 20 and 30 cycles till you get a large amount of DNA segment of our interest. That's how we can get higher amounts of PCR product and high concentration of the PCR product. Now one thing I will make sure and think one concept is very important. At the beginning of all this reaction, we need to prepare a mix. Inside this master mix, we add everything. We add our host DNA, we add our primer, we add our polymerase. We also add our nucleotide sequences, dNTPs, right? We add all these things. Now remember for this extension process, we need to carry it out at 72 degree Celsius temperature. But one thing you must understand in this particular region that normal cellular DNA polymerase cannot act properly in the 72 degree Celsius temperature because this temperature is pretty high for this to act on. So what we need to do we need to provide some heat resistant polymerase at this 72 degree Celsius temperature extension and the polymerase we utilize is derived from the bacteria called thermos aquaticus and the polymerase is called Taq polymerase or Taq polymerase it is called Taq polymerase. There are also other sources of polymerase nowadays are taken but Taq polymerase is pretty important in this reaction it is pretty efficient in this reaction and it is very much costly so we provide everything at the beginning of the reaction make the mix we just set them into a bath we can say it's a better version of a water bath heating chamber it's called a thermocycler which controls the temperature variations because we know the process will go on it's like its natural form denaturation then annealing then again nucleotidization polymerization everything will be carried out in its natural way only we need to change and vary the temperature with time gap and time and temperature is very very important in this pcr i repeat this time and temperature is very very much important You may think this 94 degree Celsius temperature, lower down it into 90 degree Celsius temperature, it will have a devastating effect, it will have a devastating change in the product yield of your PCR. So, this temperatures and time limit is very very important. And everything can be set onto the machine called thermocycler, where you just program the machine at the very beginning of your reaction, everything will be set, everything will be provided, the data will be fed onto this thermocycler. Then you make a mixture of all these things in PCR tubes, small tubes, carrying 50 microlitre maximum of this culture solution, of this master mix. And you provide it, you put it and then start the reaction. Okay. And it will automatically start the reaction. Suppose you want to carry out this reaction cycles for 30 cycles. You program this process machine to stop this process after 30 cycles. So it will carry out until 30 cycles. When the 30 cycles will be done, it will automatically switch off the reaction. Then you take out the product and it's done. It's that much simple, but you need to understand the mechanism which is very very interesting. Okay, so again, let's see at 94 degree Celsius temperature denaturation is done. Then annealing at 54 degree Celsius temperature primer start to bind. Then the extension at 72 degree Celsius temperature and the extension will be completed. Again denaturation. You can see denaturation here and these dots are annealing of the primer. Then these dots will come and these are the nucleotide sequences extension at 72 degree Celsius temperature. Okay. So how are the functions of replication achieved during the PCR? Because we need to maintain some functionality like the DNA polymerization and also denaturation and also ligation or annealing. Now melting of the DNA is done established using heat. Usually in normal process of DNA replication it is carried out by the helicase, SSB protein and topoisomerase. So we are bypassing this tape activity of these three important enzymes with only one thing which is heat in this case. So it's pretty much effective, right? Second thing is that we need to have a polymerization of the DNA. Now the polymerization of the DNA is carried out in normal cells by the DNA polymerase which is heat labile. sorry heat sensitive. Now in case of PCR we give a Taq polymerase which is not heat sensitive which is heat labile okay heat resistant type. Now the second thing is the providing the primer. The primer is provided in case of normal cellular process of DNA replication by the primers enzyme in this case we don't need primers instead we need to supply primers from the outside. The primers we design for destined to bind with the particular region flanking our interested gene. Okay. And we provide it in the reaction mix. Now the joining task in normal cell, the joining task is achieved via the ligase. In this case we don't put any kind of ligase, the fragments are short and we don't need this kind of joining because after the production of it's only only say we start most of the time we amplify the segments like 200 BP 100 150 and small sequences like that and tack polymers is pretty good at Polymerizing that amount of sequence at a time, right? So you don't require any kind of joining because any kind of replication or blocking or release is not done in this particular case. Now this is the overview again. So this is our say gene of interest. It is separated. Both the black strands are the parental strands. Now these blue strands are newly synthesized using PCR. Then again they are separated. Black, blue separated. Reds are the new one. Then again black, red separated. Oranges are the new one. And that's how we can see from 1 to 2, from 2 to 4, from 4 to 8, from 8 to 16, from 16 to 32 and so on. It will move on like the formula, say 2 to the power n. Right? So, it's giving us a logarithmic approach of the growth. So, if we make a graph of it, we can see, theoretically, it must go on and on like that. But actually, we are going to see that after 30 and 40 cycles, it will actually levels off like that. Why? We are going to see it later. It is called the Pléthiav effect. Okay. So again these are the PCR ingredients. We need to provide primers, DNA template, Taq polymerase, buffer. It must contain magnesium chloride and also potassium chloride. These salts are very very important in buffer. We need to provide dNTPs. Okay, so we need to provide all the mixtures. We not only need to provide the mixtures, we need to maintain the concentration of mixture in appropriate amount to get a better result. Okay, now let us look at the step-by-step analysis of PCR reaction. and how it is conducted. Okay, let's begin. So here says this is our DNA of interest. What we will do now and we will, during this PCR reaction we will also see the graph of going with the temperature graph, right. So temperature in y-axis time in x-axis, we can see the variation in temperature with time during the process. So this is our desired DNA. First is the 94 degree Celsius temperature melting and the melting is done utilizing heat. After that, we need to lower the temperature to 50 degree Celsius temperature for annealing of primers. Now primers come and anneal with this region and our desired region is this grey colored region in both the case. Then the extension will be carried out in 72 degree Celsius temperature in both the cases. And again melting, so this one cycle is done at this particular stage. Now this is the second cycle, again melting at 94 degree Celsius temperature. Now again after melting in 94, we are again recycling it, annealing at 50 degree Celsius temperature and extension at 72 degree Celsius temperature. This is annealing again, you can see. Then again melting at 94 degree Celsius temperature, annealing at 50 degree Celsius temperature and extension at 72 degree Celsius temperature. and it will be carried out so. So we get the fragments of defined length in our hand because at the very beginning what we produce this kind of segments at the very beginning you can see. But these are not exactly our desired segment because exactly our desired segments are this particular stretch. So once after 2 and 3 cycles of the reaction we start to get our desired product, the exact desired product. At the very beginning first 2, 1 or 2 steps we haven't got any kind of our exactly desired product because we are having extra overhangs in all the directions so once after this particular stage is reached after this particular step it will amplify that this desired product in higher and higher amounts in 2 to the power n this is the formula of amplification so sooner this unwanted DNA segments will become less and we get more and more fragments of our interest So, what it actually means, more cycles means more DNA, right? So, as we go more cycles, we produce more DNA. So, you can see markers and you can see the amplification, the amount of DNA is getting increased as we increase the number of cycles. This is the number of cycles, 0, then number of cycles, 10, very faint band, 15, little, then 20, 25, 30, after 30 cycles, we get pretty broad band, right? It is suggesting that more cycles means more DNA. Now the typical PCR mix components are made up with template DNA it is to be provided, it is variable actually to be provided to 5 to 200 nanogram then 1 millimole dNTPs 10 microliter if we need to make the total volume of 50 microliter this is a protocol you must if you are carried out this PCR reaction day to day and on and on you should have memorized all these things but I won't encourage you now to memorize but for your understanding purpose you must know template must be there, dNTPs must be there and also for the buffer we need water and buffer solution which must be provided as these are the 10x buffers we need to dilute it with water and we need to provide magnesium chloride which is playing a very vital role we are going to see it later we need to provide the forward and reverse primer and also the DNA polymerase which is the Taq polymerase right in 1.5 units so this unit is very important right okay so now let us talk about the PCR optimization in different ways So first is the buffers. Most buffers have only KCl and trees in it. Now KCl facilitates the primer binding but concentrations higher than 50 millimolar can inhibit the binding or inhibit the activity of Taq polymerase. So we need to be very careful but KCl is important because it facilitates the primer binding or the annealing. Okay, sometimes also you can see the presence of different types of detergents like Twin 20, Triton X100 and all these things. in the PCR reaction it actually enhance the specificity of the reaction and also it ensures the proper binding and specific binding of the primers to the desired sequence. Second is the concentration and application of magnesium chloride it is required for the primer binding and also it is required for the attachment of primer DNA and and the concentration of primer template association is very very important because it also importance it also are affect the product specificity and also the enzyme activity and fidelity. So these things are taken care of. So the excess of this magnesium can give a non-specific binding and the too little magnesium can give a reduced yield. So need to be very careful choosing the concentration of MgCl2 because excess of it will give non-specific binding and the less of it will give a reduced yield. Now what is the actual mechanism is that There are nucleotide sequences, the DNTPs and also templates and primers and the sequester and the chelate is magnesium ion. So you need to be very careful about the concentration of NGCl2. Third thing is the primer design. Now primer must have some important criterias. I'll be making a different video about how to select primers and what are the criterias to look for before selection of the primer design. Now this primers should be specific. against a particular desired segment, to destent against binding with a particular segment of DNA, it must have the length between 18 to 30 nucleotide sequences. The annealing temperature is from 50°C to 70°C. Though it's written from 50°C to 70°C, the actual temperature varies from 58°C to 60°C. Now, the GC content of the primer must not concede from 40 to 50 or 60% of GC reach. because if it is much more GC-rich then the removal of this cannot be possible and if it is less GC-rich then it may not end with a sufficient strong binding. Now the 3'end is very very critical. Now the 3'terminal is made up with this GC content. So if this GC clamp is very important, right? We want a GC or CG or any kind of GNC nucleotide sequences at the 3'terminal because it will ensure the proper binding and it will ensure the proper initialization of the process right and the self-complementarity must be less because if the primers are having self-complementarity for example say this is the primer if it is having self-complementarity so what it can do it can make a intra strand linkage like that interest and bond like that it can make problems right so three prime complementarity is an important fact and also C2-4 bases similar to other primers must not be there. Okay, so these things must be present in all these cases. Okay, so we need to think about these concepts before designing the primer. Now the fourth part is the cycling conditions. First thing is the denaturation. Some tack polymer is required initial denaturation which is a hot start. So we need to provide them the hot start. Sorry, we need to provide them the hot start. So we need to provide them the hot start. So at the very beginning we need to provide them with higher temperature like 94 degree celsius temperature and so. And second thing is the annealing temperature. Annealing temperature must be less than 5 degree celsius of the TM or the melting temperature of the primers. Right. So suppose this is our host DNA and. Say this is our primer is attached to the host DNA. Now there must be a melting temperature of this primer DNA conjugate. Okay, so we must provide less than 5 degree Celsius temperature of less than 5 degree Celsius temperature of the melting temperature of this conjugate, right? This is a melting temperature Tm is a melting of this DNA primer hybrid. Now you need to provide some temperature which is less than 5 degree Celsius. If If we increase this temperature, if this melting temperature of DNA primer hybrid and the annealing temperature remains the same, then it will not ensure proper binding of primer with our host DNA. So we need to be very careful about all these things. So before ordering any kind of primer and looking for any kind of primer, we must understand the melting temperature of that primer with the DNA. Decrease in annealing temperature result in non-specific binding. This is the first thing. it results in non-specific binding and increase in alanine temperature is a rest result in the reduced yield okay so decrease in alanine temperature non-specific and increase so you can see in all these cases the margin of error is very less because in all those directions if we increase the temperature a little bit or decrease the temperature a little bit it will end up with a very bad effects right so you need to maintain the temperature in a balanced way in all those directions Now the last thing is the cycle number. Now normally the cycle number theoretically can be from 50 to 40 cycles. Now, theoretical yield for this PCR reaction is 2 to the power n and we have seen it before. So, theoretically what happens this number of PCR, number of the PCR product will increase with time like a logarithmic scale like a straight line increase like that but actually it doesn't happen. because it was seen the half-life of Taq polymerase is 30 minutes at 95 degree Celsius temperature. So if you are continuously utilizing this Taq polymerase at 94 degree Celsius temperature for the 70 degree Celsius temperature for the elongation process and also we provide this temperature because remember at the very beginning, start of the very beginning the Taq polymerase are in this mixture and at the very beginning we use 94 degree Celsius temperature for denaturation of the DNA. At this 94 degree Celsius temperature, tack polymerase are still in our master mix. So, it is forced to leave in this 94 degree Celsius temperature for each 1 minute during the cycle. So, after 30 cycles, this tack polymerase will be no longer functional. Not actually no longer functional, but the functional efficiency of this tack polymerase will go down dramatically. So, as a result of this loss of efficiency of tack polymerase, we will result... in fall of the amplification product of PCR and we get a almost straight line or stationary phase reaching after the 30 cycle. So until the 30 cycle the product increase from the 28 cycle and 26 cycle to 30 cycle we are going to see a logarithmic increase but after that it will stop increasing. So this is called the Pletcher effect of PCR amplification okay so in summary Primer length should not exceed 30 N sequences. The Tm not more than 60 degree Celsius. We need to be very careful because annealing temperature is 50 and something like that. So, it must have at least 5 degree Celsius gap between them. Then, Gc content should be in the range of 40 to 60 percent. And also, if G or C is having in the C prime end, it ensures a better binding because this prevents the breathing of ends. and increases the efficiency of priming. So these are the fundamentals of PCR and the applications of PCR are enormous. We are going to see that the applications of PCR are actually everywhere in the molecular biology techniques. So we can have this molecular identification, sequencing and genetic engineering. All these cases PCR is applicable. Like the, say for example, prenatal diagnosis, mutation screening, drug discovery, genetic matching. In all these cases, we have seen the use of PCR. In genetic engineering, production of site-directed mutagenesis and gene expression studies PCR is utilized. In sequencing, bioinformatics and genome cloning and also in human genome project, PCR was a huge key thing to play. So, in all these parts are very important and utilizing PCR we can see in different molecular biology techniques the clear state of art techniques like PFG, RFLP, replication PCR, RAPD, CFLP, AFLP and sequencing. In all these cases we have seen the presence of PCR in all these different processes. In all the processes PCR is an important step. It is playing a very very important part. So, molecular biology is a nothing except for PCR nowadays because it is a key in molecular biology technique. So, I hope this video is helping you to understand PCR. Thank you.

Hello and welcome back friends. In this video we will be talking about PCR or polymerase chain reaction. It is actually better known as PCR. Let's begin.

As the name suggests, polymerase chain reaction, it means, let me discuss it to you. Polymerase means for this process we must have needed the polymerase enzyme, remember? the effect of polymerase enzyme during the DNA replication process it can add nucleotide sequences one by another and can elongate and can produce a nucleic acid chain like DNA or RNA. Now the polymerase can be of DNA polymerase and RNA polymerase both the case it can polymerize nucleotide sequences chain reaction that means the reaction procedures or sequential events in the reaction which will go on and on and on like a chain. So we are involving a polymerase enzyme for adding nucleotide sequences and making a long chain nucleotide sequences and the process will be a chain reaction process.

That means we will amplify something. We are amplifying something with this chain reaction. Whenever we are talking about a kind of chain reaction, it means we are amplifying something. Now in this case also we are amplifying.

The basic goal of utilizing polymerase chain reaction is to produce a copy of the segment of DNA in much more higher amount in high concentration. For example, say in this whole DNA content, this red colored segment of this gene is of our interest. This is the wanted gene. Now we want to amplify the number of this gene for our purpose, for our experimental purpose.

So utilizing this polymerase chain reaction is allowing us to increase the number of that particular region of this DNA not the other regions of the DNA. And we can see after many cycles so it's a repeated process as you see in the name chain reaction so it is increasing the number of this template DNA of our interest in chain reaction or in higher magnitude in greater number as we go on in logarithmic spell and what we can get from one template of the DNA 2 then 2 to 4 then 4 to 8 and then 8 to 16 and so on it will start to generate a lot of number of this particular template DNA of our interest after many different cycles. This is the importance of PCR for the amplification of a particular segment inside the DNA.

Suppose a desired region of our interest which is a gene a particular gene. It's an exponential amplification process. Now for this PCR reaction there are several steps of this pci reaction actually we can divide into three major steps of this pci reaction now it's all about the simple or basic mechanism of dna maturation dna naturation and denaturation okay dna naturation and denaturation is playing the important role here and also simple polymerization process that means the the dna polymerization process is also important so if you understand the dna polymerization process and denaturation of the dna you can understand this process pretty easily For example, say this is our desired DNA. Somewhere in between this desired DNA, we need to copy the gene. Right?

So, suppose from this part to this part, we need to copy this gene. So, what we'll do, we will amplify this segment of this gene, but not amplifying the rest of the part. So, what we'll do in this case, the first step is the denaturation process. To denature the complementary strands of a DNA, we must heat it.

So, we'll do that. at 94 degree Celsius temperature for about one minute. Now heating it in 94 degree Celsius temperature for one minute actually 90 or above 90 degree Celsius temperature for one minute it will denature the DNA and we are having this DNA in our hand. Now this is a host DNA and one thing you need to make sure before the denaturation or setting this denaturation temperature is that what is the Tm or melting temperature of the DNA.

We know the melting temperature of the DNA varies from one species to another species. Right. Because the constituents of this nucleic acid can be varied. So the number of GC is very very important.

GC content is very very important. We know this guanosine added with cytosine via the C hydrogen bonds. So they are much more stronger bonds. So we need to give much more energy to break GC bonds than to break the AT bonds.

So that's why. we need to use high amount of temperature to break those DNAs or to separate those DNA strands which are having more GC than 80, right? So that's why we need to be very careful about the construction of the DNA.

We need to look for the nucleotide makeup of the DNA before setting this temperature. Now let's say we set the temperature in this case 94 degree Celsius then what it will do? It will denature the DNA in a hole. After denaturing the DNA two strands are separated. So this is strand 1, this is strand 2. After this strand separation what we'll do is start providing some important oligonucleotide sequences.

Stretch of oligonucleotide sequences. Why? Why we require this stretch of oligonucleotide sequences? Because remember what we need to do, we need to do a polymerase reaction, a polymerization reaction. And we know that DNA polymerase is only function, it can only function when we provide it a 3 prime hydroxyl.

These are the basics of DNA replication process. I know, I hope you understand these things. If you don't just go back and look for the video of DNA replication and DNA polymerization videos in my website you can find it. Now these DNA polymers must have the C-Pyme hydroxyl to continue the polymerization events to provide it during the DNA replication also we need to attach some small sequence of primer.

Now in this case also need to provide some oligonucleotide stretch which is called primer. Now we need to provide this primer and this primer will attach to this DNA segments flanking the region of our interest. Suppose this is the region of our interest.

So, if we attach the primer flanked both the ways of our desired region of our interest. Okay, we give this primer. But one thing you need to make sure is that the primer sequences that we provide must have at least greater than 60% sequence similarities sequence complementarity, not similarity sequence complementary with the host DNS trans.

otherwise it will not be able to bind with the host strand, right? So, we need to make sure this thing, okay? And also for the binding of the primer or for the annealing of the primer with the host DNA, we must lower down the temperature from 94 degree Celsius temperature to 45, sorry, to 54 degree Celsius temperature. And we allow this annealing process to happen in 45 minutes to, sorry, 45 seconds to 50 seconds like that, okay?

So we require two different primers. One primer is called the forward primer. Another primer is called the reverse primer. Now in this case we attach, why is called forward and reverse?

We know the polymerization step carries out from the 5'end to the 3'end. So those primers which are designed and attached to the 5'end to 3'end direction will be called the forward primers. For example, let me change the color here. Sorry, yeah.

This one is a forward primer because it is testing to 5 prime end to 3 prime end and those primers which are getting from 3 prime end to 5 prime end the opposite direction it will be called the reverse primer So we get both the primers in this account We are getting forward primers as well as reverse primers Altogether we need to lower the temperature to the 50 to 54 degree Celsius temperature We need to allow it to happen for 45 seconds and then this primer will anneal with the host DNA sequence. Now after the anneal, remember the two strands are separated and we add primers both the strands. So utilizing both the DNA strands for amplification of our desired gene.

Now after that we must add this all this nucleotide sequences there A, G, T and C because we require this nucleotide sequences to be attached with each other by the DNA polymerase. So nucleotide sequences will be attached using DNA polymerase. and we need to again rise the temperature a little bit to 72 degree Celsius temperature and this DNTPs will keep on binding and we need to carry out this for 2 minutes Okay, so this is the total process First denaturation in 94 degree Celsius temperature for 1 minute Second is annealing of primers at 54 degree Celsius temperature for 45 seconds to bind the forward and reverse primers After the binding of primers, we will provide the polymerase as well as the dNTPs to attach them and to polymerize these nucleotide sequences at 72 degrees Celsius temperature for 2 minutes and it will extend the primers and it will produce the segment of our interest or the gene of our interest. And this is called a one cycle of a polymerase chain reaction. So a denaturation, annealing and a renaturation and an extension, sorry, this whole procedure.

is called the one cycle of this PCR or polymerase chain reaction. Now we keep on repeating these cycles to keep on repeating and producing our desired gene segments and amplifying our desired gene segments almost like 20 and 30 cycles till you get a large amount of DNA segment of our interest. That's how we can get higher amounts of PCR product and high concentration of the PCR product. Now one thing I will make sure and think one concept is very important.

At the beginning of all this reaction, we need to prepare a mix. Inside this master mix, we add everything. We add our host DNA, we add our primer, we add our polymerase. We also add our nucleotide sequences, dNTPs, right? We add all these things.

Now remember for this extension process, we need to carry it out at 72 degree Celsius temperature. But one thing you must understand in this particular region that normal cellular DNA polymerase cannot act properly in the 72 degree Celsius temperature because this temperature is pretty high for this to act on. So what we need to do we need to provide some heat resistant polymerase at this 72 degree Celsius temperature extension and the polymerase we utilize is derived from the bacteria called thermos aquaticus and the polymerase is called Taq polymerase or Taq polymerase it is called Taq polymerase. There are also other sources of polymerase nowadays are taken but Taq polymerase is pretty important in this reaction it is pretty efficient in this reaction and it is very much costly so we provide everything at the beginning of the reaction make the mix we just set them into a bath we can say it's a better version of a water bath heating chamber it's called a thermocycler which controls the temperature variations because we know the process will go on it's like its natural form denaturation then annealing then again nucleotidization polymerization everything will be carried out in its natural way only we need to change and vary the temperature with time gap and time and temperature is very very important in this pcr i repeat this time and temperature is very very much important You may think this 94 degree Celsius temperature, lower down it into 90 degree Celsius temperature, it will have a devastating effect, it will have a devastating change in the product yield of your PCR.

So, this temperatures and time limit is very very important. And everything can be set onto the machine called thermocycler, where you just program the machine at the very beginning of your reaction, everything will be set, everything will be provided, the data will be fed onto this thermocycler. Then you make a mixture of all these things in PCR tubes, small tubes, carrying 50 microlitre maximum of this culture solution, of this master mix.

And you provide it, you put it and then start the reaction. Okay. And it will automatically start the reaction. Suppose you want to carry out this reaction cycles for 30 cycles. You program this process machine to stop this process after 30 cycles.

So it will carry out until 30 cycles. When the 30 cycles will be done, it will automatically switch off the reaction. Then you take out the product and it's done.

It's that much simple, but you need to understand the mechanism which is very very interesting. Okay, so again, let's see at 94 degree Celsius temperature denaturation is done. Then annealing at 54 degree Celsius temperature primer start to bind.

Then the extension at 72 degree Celsius temperature and the extension will be completed. Again denaturation. You can see denaturation here and these dots are annealing of the primer.

Then these dots will come and these are the nucleotide sequences extension at 72 degree Celsius temperature. Okay. So how are the functions of replication achieved during the PCR?

Because we need to maintain some functionality like the DNA polymerization and also denaturation and also ligation or annealing. Now melting of the DNA is done established using heat. Usually in normal process of DNA replication it is carried out by the helicase, SSB protein and topoisomerase. So we are bypassing this tape activity of these three important enzymes with only one thing which is heat in this case. So it's pretty much effective, right?

Second thing is that we need to have a polymerization of the DNA. Now the polymerization of the DNA is carried out in normal cells by the DNA polymerase which is heat labile. sorry heat sensitive.

Now in case of PCR we give a Taq polymerase which is not heat sensitive which is heat labile okay heat resistant type. Now the second thing is the providing the primer. The primer is provided in case of normal cellular process of DNA replication by the primers enzyme in this case we don't need primers instead we need to supply primers from the outside. The primers we design for destined to bind with the particular region flanking our interested gene. Okay.

And we provide it in the reaction mix. Now the joining task in normal cell, the joining task is achieved via the ligase. In this case we don't put any kind of ligase, the fragments are short and we don't need this kind of joining because after the production of it's only only say we start most of the time we amplify the segments like 200 BP 100 150 and small sequences like that and tack polymers is pretty good at Polymerizing that amount of sequence at a time, right? So you don't require any kind of joining because any kind of replication or blocking or release is not done in this particular case.

Now this is the overview again. So this is our say gene of interest. It is separated.

Both the black strands are the parental strands. Now these blue strands are newly synthesized using PCR. Then again they are separated. Black, blue separated.

Reds are the new one. Then again black, red separated. Oranges are the new one. And that's how we can see from 1 to 2, from 2 to 4, from 4 to 8, from 8 to 16, from 16 to 32 and so on.

It will move on like the formula, say 2 to the power n. Right? So, it's giving us a logarithmic approach of the growth. So, if we make a graph of it, we can see, theoretically, it must go on and on like that. But actually, we are going to see that after 30 and 40 cycles, it will actually levels off like that.

Why? We are going to see it later. It is called the Pléthiav effect. Okay.

So again these are the PCR ingredients. We need to provide primers, DNA template, Taq polymerase, buffer. It must contain magnesium chloride and also potassium chloride. These salts are very very important in buffer.

We need to provide dNTPs. Okay, so we need to provide all the mixtures. We not only need to provide the mixtures, we need to maintain the concentration of mixture in appropriate amount to get a better result. Okay, now let us look at the step-by-step analysis of PCR reaction. and how it is conducted.

Okay, let's begin. So here says this is our DNA of interest. What we will do now and we will, during this PCR reaction we will also see the graph of going with the temperature graph, right. So temperature in y-axis time in x-axis, we can see the variation in temperature with time during the process.

So this is our desired DNA. First is the 94 degree Celsius temperature melting and the melting is done utilizing heat. After that, we need to lower the temperature to 50 degree Celsius temperature for annealing of primers. Now primers come and anneal with this region and our desired region is this grey colored region in both the case.

Then the extension will be carried out in 72 degree Celsius temperature in both the cases. And again melting, so this one cycle is done at this particular stage. Now this is the second cycle, again melting at 94 degree Celsius temperature. Now again after melting in 94, we are again recycling it, annealing at 50 degree Celsius temperature and extension at 72 degree Celsius temperature.

This is annealing again, you can see. Then again melting at 94 degree Celsius temperature, annealing at 50 degree Celsius temperature and extension at 72 degree Celsius temperature. and it will be carried out so.

So we get the fragments of defined length in our hand because at the very beginning what we produce this kind of segments at the very beginning you can see. But these are not exactly our desired segment because exactly our desired segments are this particular stretch. So once after 2 and 3 cycles of the reaction we start to get our desired product, the exact desired product. At the very beginning first 2, 1 or 2 steps we haven't got any kind of our exactly desired product because we are having extra overhangs in all the directions so once after this particular stage is reached after this particular step it will amplify that this desired product in higher and higher amounts in 2 to the power n this is the formula of amplification so sooner this unwanted DNA segments will become less and we get more and more fragments of our interest So, what it actually means, more cycles means more DNA, right? So, as we go more cycles, we produce more DNA.

So, you can see markers and you can see the amplification, the amount of DNA is getting increased as we increase the number of cycles. This is the number of cycles, 0, then number of cycles, 10, very faint band, 15, little, then 20, 25, 30, after 30 cycles, we get pretty broad band, right? It is suggesting that more cycles means more DNA. Now the typical PCR mix components are made up with template DNA it is to be provided, it is variable actually to be provided to 5 to 200 nanogram then 1 millimole dNTPs 10 microliter if we need to make the total volume of 50 microliter this is a protocol you must if you are carried out this PCR reaction day to day and on and on you should have memorized all these things but I won't encourage you now to memorize but for your understanding purpose you must know template must be there, dNTPs must be there and also for the buffer we need water and buffer solution which must be provided as these are the 10x buffers we need to dilute it with water and we need to provide magnesium chloride which is playing a very vital role we are going to see it later we need to provide the forward and reverse primer and also the DNA polymerase which is the Taq polymerase right in 1.5 units so this unit is very important right okay so now let us talk about the PCR optimization in different ways So first is the buffers. Most buffers have only KCl and trees in it.

Now KCl facilitates the primer binding but concentrations higher than 50 millimolar can inhibit the binding or inhibit the activity of Taq polymerase. So we need to be very careful but KCl is important because it facilitates the primer binding or the annealing. Okay, sometimes also you can see the presence of different types of detergents like Twin 20, Triton X100 and all these things. in the PCR reaction it actually enhance the specificity of the reaction and also it ensures the proper binding and specific binding of the primers to the desired sequence. Second is the concentration and application of magnesium chloride it is required for the primer binding and also it is required for the attachment of primer DNA and and the concentration of primer template association is very very important because it also importance it also are affect the product specificity and also the enzyme activity and fidelity.

So these things are taken care of. So the excess of this magnesium can give a non-specific binding and the too little magnesium can give a reduced yield. So need to be very careful choosing the concentration of MgCl2 because excess of it will give non-specific binding and the less of it will give a reduced yield.

Now what is the actual mechanism is that There are nucleotide sequences, the DNTPs and also templates and primers and the sequester and the chelate is magnesium ion. So you need to be very careful about the concentration of NGCl2. Third thing is the primer design. Now primer must have some important criterias. I'll be making a different video about how to select primers and what are the criterias to look for before selection of the primer design.

Now this primers should be specific. against a particular desired segment, to destent against binding with a particular segment of DNA, it must have the length between 18 to 30 nucleotide sequences. The annealing temperature is from 50°C to 70°C.

Though it's written from 50°C to 70°C, the actual temperature varies from 58°C to 60°C. Now, the GC content of the primer must not concede from 40 to 50 or 60% of GC reach. because if it is much more GC-rich then the removal of this cannot be possible and if it is less GC-rich then it may not end with a sufficient strong binding.

Now the 3'end is very very critical. Now the 3'terminal is made up with this GC content. So if this GC clamp is very important, right? We want a GC or CG or any kind of GNC nucleotide sequences at the 3'terminal because it will ensure the proper binding and it will ensure the proper initialization of the process right and the self-complementarity must be less because if the primers are having self-complementarity for example say this is the primer if it is having self-complementarity so what it can do it can make a intra strand linkage like that interest and bond like that it can make problems right so three prime complementarity is an important fact and also C2-4 bases similar to other primers must not be there. Okay, so these things must be present in all these cases.

Okay, so we need to think about these concepts before designing the primer. Now the fourth part is the cycling conditions. First thing is the denaturation. Some tack polymer is required initial denaturation which is a hot start.

So we need to provide them the hot start. Sorry, we need to provide them the hot start. So we need to provide them the hot start.

So at the very beginning we need to provide them with higher temperature like 94 degree celsius temperature and so. And second thing is the annealing temperature. Annealing temperature must be less than 5 degree celsius of the TM or the melting temperature of the primers.

Right. So suppose this is our host DNA and. Say this is our primer is attached to the host DNA. Now there must be a melting temperature of this primer DNA conjugate.

Okay, so we must provide less than 5 degree Celsius temperature of less than 5 degree Celsius temperature of the melting temperature of this conjugate, right? This is a melting temperature Tm is a melting of this DNA primer hybrid. Now you need to provide some temperature which is less than 5 degree Celsius. If If we increase this temperature, if this melting temperature of DNA primer hybrid and the annealing temperature remains the same, then it will not ensure proper binding of primer with our host DNA. So we need to be very careful about all these things.

So before ordering any kind of primer and looking for any kind of primer, we must understand the melting temperature of that primer with the DNA. Decrease in annealing temperature result in non-specific binding. This is the first thing.

it results in non-specific binding and increase in alanine temperature is a rest result in the reduced yield okay so decrease in alanine temperature non-specific and increase so you can see in all these cases the margin of error is very less because in all those directions if we increase the temperature a little bit or decrease the temperature a little bit it will end up with a very bad effects right so you need to maintain the temperature in a balanced way in all those directions Now the last thing is the cycle number. Now normally the cycle number theoretically can be from 50 to 40 cycles. Now, theoretical yield for this PCR reaction is 2 to the power n and we have seen it before. So, theoretically what happens this number of PCR, number of the PCR product will increase with time like a logarithmic scale like a straight line increase like that but actually it doesn't happen.

because it was seen the half-life of Taq polymerase is 30 minutes at 95 degree Celsius temperature. So if you are continuously utilizing this Taq polymerase at 94 degree Celsius temperature for the 70 degree Celsius temperature for the elongation process and also we provide this temperature because remember at the very beginning, start of the very beginning the Taq polymerase are in this mixture and at the very beginning we use 94 degree Celsius temperature for denaturation of the DNA. At this 94 degree Celsius temperature, tack polymerase are still in our master mix. So, it is forced to leave in this 94 degree Celsius temperature for each 1 minute during the cycle.

So, after 30 cycles, this tack polymerase will be no longer functional. Not actually no longer functional, but the functional efficiency of this tack polymerase will go down dramatically. So, as a result of this loss of efficiency of tack polymerase, we will result... in fall of the amplification product of PCR and we get a almost straight line or stationary phase reaching after the 30 cycle.

So until the 30 cycle the product increase from the 28 cycle and 26 cycle to 30 cycle we are going to see a logarithmic increase but after that it will stop increasing. So this is called the Pletcher effect of PCR amplification okay so in summary Primer length should not exceed 30 N sequences. The Tm not more than 60 degree Celsius. We need to be very careful because annealing temperature is 50 and something like that. So, it must have at least 5 degree Celsius gap between them.

Then, Gc content should be in the range of 40 to 60 percent. And also, if G or C is having in the C prime end, it ensures a better binding because this prevents the breathing of ends. and increases the efficiency of priming. So these are the fundamentals of PCR and the applications of PCR are enormous. We are going to see that the applications of PCR are actually everywhere in the molecular biology techniques.

So we can have this molecular identification, sequencing and genetic engineering. All these cases PCR is applicable. Like the, say for example, prenatal diagnosis, mutation screening, drug discovery, genetic matching.

In all these cases, we have seen the use of PCR. In genetic engineering, production of site-directed mutagenesis and gene expression studies PCR is utilized. In sequencing, bioinformatics and genome cloning and also in human genome project, PCR was a huge key thing to play. So, in all these parts are very important and utilizing PCR we can see in different molecular biology techniques the clear state of art techniques like PFG, RFLP, replication PCR, RAPD, CFLP, AFLP and sequencing. In all these cases we have seen the presence of PCR in all these different processes.

In all the processes PCR is an important step. It is playing a very very important part. So, molecular biology is a nothing except for PCR nowadays because it is a key in molecular biology technique. So, I hope this video is helping you to understand PCR. Thank you.

Transcript for:Understanding Polymerase Chain Reaction Techniques

Transcript for:
Understanding Polymerase Chain Reaction Techniques