 Say you want to perform a gene cloning experiment, the very first thing you would do is to extract the gene you want out from a long piece of DNA. Let's say this small part over here is the part you want to extract. This is the gene that you are interested in. So with the help of molecular caesars or restriction enzyme, you extract the gene out of that long piece of DNA and then the next step would be to put it inside of a vector. Now to insert it into a vector, we again need molecular caesars or restriction enzymes. But here the question is, where exactly in this closed loop in this vector will the restriction enzyme make the cut? And how many number of times will it make the cut? Once, twice or multiple times. So in this video, we will try and understand all about the site in which we insert the gene of interest. How restriction enzyme decides where to cut and where to insert the gene that we care about. Alright, let's begin with the first question of where exactly the restriction enzyme makes the cut. Now, if you have gone through the video of restriction enzymes, you would know that every restriction enzyme has a unique sequence that it recognizes and every time makes the cut at the same specific point. So if we have to talk about the restriction enzyme ECHO R1, let me write it down, ECHO R1. Say we want to cut the vector with this particular enzyme. So the vector should have this palindromic sequence inside of it, that is GAA TTC and it would read the same from both the directions. Now this ECHO R1 will make a cut between guanin and edinin on both the strains and the strains would separate out having two overhangs. So this brings us to the answer of the first question of where exactly in the vector the restriction enzyme would make a cut. So the answer is every time it encounters the sequence it recognizes, just like this sequence is recognized by ECHO R1, it would be some other sequence for some other restriction enzyme. So every time a restriction enzyme encounters the sequence it recognizes, it will make a cut exactly at the same point every single time. Now say we have this ECHO R1 restriction site, that means this palindromic sequence at six different places in our vector and we allow this ECHO R1 to act on our vector, what do you think would happen? It will cut the vector at six different places. So this will result in small small fragments of our plasmid or the vector and the small fragments will not have the property that we look for in a vector, right? The most important property being self-replication, the small small fragments will lose that property and therefore this small fragments will be of no use to us. Therefore it is always preferred and scientists design it that way that one vector should have just one site, one cloning site for a single restriction enzyme. So one restriction enzyme say ECHO R1 will not have more than one or two cloning sites. We call it cloning sites because we insert the gene that we want to clone inside of it. We also call restriction sites because we cut it with the restriction enzymes and we also call recognition sites because these are the sites where we find the sequence that can be recognized by the restriction enzyme. Alright now that we have cut our vector with ECHO R1, our vector will open up and would look something like this, it will have overhangs or sticky ends and they can bind to each other and can form the circular plasmid again or it also has the property to stick to any piece of DNA that has complementary base pairs as the overhangs. So how do we cut the gene of interest so that it has complementary base pairs? Let's say the restriction site that is present near to the gene of interest is not ECHO R1 but another restriction enzyme say BAMH1. Now BAMH1 will recognize another nucleotide sequence which is this one G, G, A, T, C, C and it makes a cut between two gonon on both the strands and this is how it splits up and these are the overhangs, these are the nucleotides it will have on its overhangs that is C, T, A and G. Now I want you to pause for a while and think off if these overhangs will fit perfectly into the overhangs that we created after cutting the vector with ECHO R1. Well since the overhangs do not fit each other, since they do not have complementary base pairs there is no condition under which they will ever fit. But let's assume we have a site in the gene which can be recognized by ECHO R1 restriction enzyme and what will happen if we cut the gene of interest with the same restriction enzyme? Let's go ahead and do that. And when you allow this piece of gene to go and bind to the vector they will perfectly fit, they will hug each other just like long lost best friends. So this has made it super clear that we have to cut the gene of interest and the vector with the same restriction enzyme so that they can fit each other. Now what if we have the restriction site for any random restriction enzyme say let us consider restriction enzyme X and we can cut the gene of interest only with that restriction enzyme. Now to use the same vector for that gene we will require that our vector should have, let me bring in the uncut vector. There you go. So now in order to cut the gene with X restriction enzyme we need to have a site in our vector that can be recognized by the same restriction enzyme X. Now some other day there will be the requirement to cut the gene with some other restriction enzyme say restriction enzyme Y. So we will require a site in the vector that can be recognized by that restriction enzyme. So to tell you in a nutshell a scientist will have no idea what restriction enzyme he would require to cut the next gene he would want to clone and therefore the vectors are made in such a way that it has multiple sites for different restriction enzymes so that a scientist can go ahead and insert any gene he wants because the vector will give the flexibility to cut the vector with different restriction enzymes. Let me put some more sites of restriction enzyme here. Say this is for restriction enzyme A, this here is for restriction enzyme B. Now the site in which we find all these sites for different restriction enzymes we call it the multiple cloning sites. Well this multiple cloning site in a vector is always located inside of selectable marker gene. Now why we have it inside of selectable marker gene is again a very interesting concept. We will have a whole different video on it but for now just remember that one vector have multiple sites that can be cleaved by different restriction enzymes in which we can put in the gene that we want and that this site is located inside of a selectable marker gene. Selectable marker gene. Alright now that our vector has multiple sites for different restriction enzymes you can go ahead and clone any gene you want because probably the restriction enzyme you would use to cut your gene we will have a site for that restriction enzyme in our vector. Alright now let's quickly summarize what we learnt in this video. The very first thing we learnt is about restriction enzymes and that we use the same restriction enzyme to cut the gene of interest and to cut our vector so that they have overhangs that perfectly fit each other. The next thing we learnt about is that one restriction enzyme should have one or a maximum of two cloning sites because if we have multiple sites it will make multiple fragments of our vector and we do not want that right? The third and the most important thing we learnt is about the multiple cloning sites that one vector should have multiple cloning sites so that it can accommodate genes which are cut with different restriction enzymes.