 Whenever we come down with something our first order of business is to find out exactly what it is that is making us feel So terrible. So we go to the doctor and ask them to diagnose us He they ask us about the signs and symptoms we're facing and then advise us to take some tests before they can Officially pin down the exact cause of the condition now these Tests which you which they ask us to do these tests are done to understand why We have the signs and symptoms in the first place These tests they answer questions like what is why is your stomach aching so much or what makes your muscles swell so much or Is your immune system working? Functioning properly against the condition that you're facing all these types of stuff now these Tests that we have some of them are more conventional testing methods like Analyzing the urine or blood samples and those tests will detect the pathogen But only if they are present in really large numbers Which means that the disease needs to spread up to a certain extent in your body for those pathogens to Show up in your samples in large numbers So that kind of makes these methods a bit tricky to go about and not just that more over these These conventional methods they are pretty time-consuming and they take a longer time to show the results and So they don't detect pathogens at lower concentrations and they take time So ideally it would be best if we have a method which is not just faster But it can also detect the pathogen as soon as it infects you before the disease can spread or it becomes life-threatening and That is possible with the help of something called molecular diagnostics now these methods They are so sensitive that they can detect the pathogen in the very early stages of the disease and It can do so by analyzing the molecules which are present in your tissues like your DNA or your RNA Then the proteins and fats and etc. etc. All of those molecules I'll give you an example So there of one of the methods that we see in molecular diagnostics what it does that instead of detecting the pathogen directly It detects the antibodies instead now your body will produce antibodies if the pathogen is present, right? So if these antibodies Are detected by that method then it confirms the presence of the pathogen against which these antibodies are Produced so we have a bunch of different things categorized under molecular diagnostics and one of the most common methods is PCR or Polymerase chain reaction now you must have heard this term PCR quite a number of times during the whole Covid era PCR or polymerase chain reaction like I mentioned is this method that has the ability to Amplify or increase the amount of nucleic acid that aka the DNA or the RNA of a bacteria or a virus so when the disease is at its early stage even when the Concentration of the bacteria or the virus is really really low PCR will find the nucleic acid of that pathogen and Amplify it and it is this amplification Which makes it easier or allows us to detect the pathogen pretty easily But the question is that how is PCR amplifying the DNA or the RNA of the Vi of the pathogen how exactly is it doing that now some of you may already know the Mechanism behind it, but let's do a quick rundown of it anyway Let's say that we want to amplify the COVID virus RNA so this is our COVID virus RNA now to make PCR work. We're gonna need four different things We're gonna start off with a double-stranded DNA template Then we need four different types of nucleotides so you can see there are four types over here different colors and Then after that we need a DNA polymerase enzyme, you know the enzyme that Helps that adds nucleotides to the DNA that enzyme and then finally we need primers But wait a second. I just told you that we're gonna need some DNA to even start off with the PCR process and COVID virus has RNA. So what are we gonna do about that? Well, we're gonna use an enzyme called reverse Transcriptase now what this enzyme is going to do is that it will convert this RNA Into the double-stranded DNA. So once we have our DNA with us, we can proceed with the rest of the PCR process Now we know that what is the purpose of the DNA like we need the DNA to start off But what about these other things? What what do the rest of these things do? Let's take a much closer look at the process now Alright, so we have our DNA over here And the first thing that we're gonna do is that we'll be separating the strands of this double-stranded DNA with the help of Heat so if you provide enough heat somewhere around 95-ish degree Celsius and if you provide this heat then what's gonna happen is that these strands They will start coming apart or they will denature and they will separate out something like this Now the second step that what we're gonna do is that we're gonna let the primers attached to these separated strands of DNA now primers are these Okay, let's bring in our primers first these are the primers and they have attached to their complementary Sections on the viral DNA and they have bound with them and primers are essentially a short pieces of DNA and Like I said, they find the complementary basis on this viral DNA and attached to them So the DNA strands separate primers come in they bind and they attach themselves to their complementary basis on the viral DNA The third step or the last step is all about the DNA polymerase enzyme So what it's going to do it's going to attach itself to the three prime end of the primer and then start adding All the nucleotides to this end constantly so new strands will now be formed against these separated strands and we will have an increased number of DNA that we didn't have before so we started off with one double-stranded DNA and by the end of this one cycle we have two so this entire cycle from Separating out the strands to adding the primers then DNA polymerase Adding the nucleotides and making new strands this entire cycle is repeated several times till we have a huge number of DNA or huge number of nucleic acid with us So this will keep on increasing so from one cycle we get to the next will get four Then other the next one will get eight or so on and so forth. So this is how it exponentially increases the amount of Nucleic acid aka amplification of DNA, but how does any of this matter anyway, right? I mean, yes, we do have a lot of DNA with us But how are we going to determine what pathogen it is or confirm the presence of a pathogen from just their DNA? Like how are we supposed to we can't see anything we can't visualize anything so how are we supposed to a Conclude that whether there's a pathogen here or not Well, we do have actually and we do have a way of visualizing our results over here And we do that with the help of yet another method called gel electrophoresis So let's make some more space and This is gel electrophoresis more space All right, so we use gel electrophoresis to visualize the results that we get from the PCR machine Now, how does that happen? Gel electrophoresis is a method that separates out DNA based on their size using electricity It consists of this slab of gel which has these wells over there So do you see these wells these are the wells which are present in this lab itself in one of the wells Let's say that we add our DNA the amplified DNA that we have and in the other well we have added our marker DNA So this are the very first column that you can see towards your left. Let's say that this is the marker DNA and This is our amplified DNA the marker DNA is kind of like a map or a standard which Which which we can compare to or refer to and find out the size of the amplified DNA Technically this marker DNA is composed or it consists of these short pieces of DNA with very specific lengths And these lengths are something that we are aware of So when you run this marker DNA on the gel electrophoresis set up Then you will see these bands being formed and each of these bands they represent a DNA piece of a very specific size The smaller ones will go or very much away from the wells and the larger the pieces of DNA They will stay closer to the wells so now this marker DNA acts as the map and we are going to compare our Amplified DNA to this marker DNA and since we already know the sequence and the length of the viral DNA all we have to do is match the band that we are getting from From this amplified in from this viral DNA to the band that we are expecting it to match on the marker DNA So let's say that this is the viral Size that we're looking for this one right here. So this is our expected Size and let's say that then this amplified DNA to runs, right? So it runs and it shows up somewhere here. So now our results match We are matching the viral DNA band to the expected band from that marker DNA So now that these two match they kind of confirm the fact that the path pathogen is present And because of this the report is now going to be a positive report that is positive for the presence of that pathogen But this is just about COVID PCR is also used to Detect a bunch of different diseases other than COVID as well like HIV and Ebola and Now if you think that you know, this makes PCR such a powerful tool Then wait till you find out about the other different methods that we have in molecular diagnostics