Lecture 5.2 Building with DNA — Compatible Ends

HAZEL SIVE: From your class exercise about restriction endonucleases, you should now be able to manipulate a piece of DNA to reveal blunt ends or sticky ends. You should know whether or not it's a 5-prime overhang or a 3-prime overhang. And this will set us up for the next topic I want to discuss, which is the question of the vector and ligation. Let's set the stage for this topic by looking back at the overall view that I gave you on cloning and using a gene of interest. We've talked about cutting the DNA now. And we haven't talked about how you exactly isolate your gene of interest. But we'll not worry about that for the moment. What we need to talk about now is the next step, how you're going to put your gene of interest into some kind of carrier DNA molecule that will allow it to replicate to high copy number. And the reason that this can occur is because of these things called vectors. A vector is really a virus that grows in bacteria. It may be pathogenic, or it may be harmless to the bacterium. The vector is made of DNA. And it is often circular, in which case it has got a special name, which is a plasmid. The trick about plasmids, and any vector, is that they have something called an origin of replication. This is abbreviated ORI. And this origin in the vector-- I'll write it out-- is an origin of replication. And this refers to DNA replication. The origin is a particular sequence in the plasmid that tells DNA replication to begin. It is a start site for DNA synthesis. In fact, it's not just plasmids and vectors that have got origins of replication. Our own chromosomes do, too. I just didn't tell you about them when we were talking about DNA synthesis. But now you've heard of them, you can add that to your compendium of knowledge about DNA synthesis in general. This origin of replication allows the plasmid to replicate within the bacterial host cell to very high copy number, 10,000 copies or more. And because you could grow billions and billions of bacteria without too much difficulty, you can land up with very large amounts of DNA-- grams, even kilograms of DNA from the right amount of bacteria that are growing the viral vector. OK. So the origin of replication allows the vector to replicate to something like 10,000 copies per cell. And that will give you lots of DNA. There's one other property that vectors usually have. And that is called a selectable marker, or two selectable markers. And I'll tell you what they are in a moment. But let's just put down that vectors also have a selectable marker. There's our vector. What we have to do now is to insert the gene of interest into the vector. We have to do a pasting. And we paste the gene of interest into the vector using a particular enzyme called DNA ligase. Our gene of interest is pasted, or covalently bonded, through phosphodiester bonds that join up the nucleic acid polymer is pasted or covalently bonded into the vector using an enzyme called DNA ligase. And just a little note-- you heard about restriction endonuclease and DNA ligase. If you hear about a molecule that's got the "-ase" at the end, it's usually an enzyme. What is this DNA ligase? It will join together any two compatible DNA ends, OK? What does that mean? So ligase joins two compatible DNA ends. And now, you have to think back to the restriction endonuclease ends. Any two blunt ends can be joined together because there's bluntness there. There's no kind of nucleic acid hybridization involved. Any two blunt ends, restriction enzyme endonuclease ends can ligate. But the same thing is not true of sticky ends. There, you've got the single stranded bits sticking out. That's why they're sticky. And in order to get sticky ends to ligate, those sticky single stranded bits have to base pair with one another. If they can base pair exactly, then ligase will join them together and make a covalently closed molecule. So two complementary, or ends that can base pair, those complementary ends can ligate. After ligation of restriction endonuclease cut DNA, you may or may not regenerate a restriction endonuclease site, OK? That's not part of the deal. So you may or may not regenerate a restriction endonuclease site. Let's look at a couple of slides, so that you can see what I mean. Here, firstly, is your basic plasmid vector. This one, called pBR322, was one of the very first that was used in genetic engineering. You can see a region called ORI. That is the origin of replication. And it's where DNA replication starts. Both the vector and the gene of interest that you insert in it will be replicated, starting at this origin of replication. And then there are these selectable markers that we mentioned, that are here listed amp and tet. Now, let's look at some compatible ends. Any blunt ends can ligate. I just made up two in the top of the slide. One of them is half of an SmaI site, but the other one is a different site. But they can ligate just fine because all you need are 3-prime and 5-prime ends to join together. You need the 3-prime hydroxl and the 5-prime phosphate. And then ligase can come along. And it can make a phosphodiester bond between those two molecules and join them back together again. Any complementary sticky ends can ligate. So complementary means that they can base pair. So for example, in the example of the EcoRI site, you can see that if you take two 1/2 EcoRI sites and put them back together again, that they will be able to ligate and regenerate a covalently closed DNA molecule and regenerate an EcoRI site, OK? The base pairing is exact there. The AATT on this molecule will base pair with the TTAA on that molecule. And you will get out the perfect covalently closed DNA. On the bottom of the slide, you'll see that there are two compatible ends there. They have, again, got the AATT TTAA base pairing that can make a closed DNA molecule that ligase can work with. But in fact, the outcome is not a restriction site. It's not an EcoRI site. There is an AT on the 5-prime end of the top strand, and then the 3-prime end of the bottom strand. And that doesn't give you an EcoRI site, OK? So that's some detail about compatible ends. And you will need to be able to work with those in order to figure out whether or not your gene of interest can ligate into your vector. Let's complete the process of thinking about now how to get the gene of interest into the vector, OK? And the way it goes is that you have to get your vector ready to receive your gene of interest. Usually, you cut your vector with the same restriction enzyme or enzymes that you've cut your gene of interest with. And then the ends can go together in a matching way. And you'll get a covalently closed recombinant DNA molecule that has got both your vector and your gene of interest. So you're going to prepare a vector. And you'll cut it with a restriction enzyme or restriction enzymes the same as used to cut the gene of interest. You then mix together your cut vector and your cut gene of interest. And they should have, at this point, compatible ends. That is your job as a genetic engineer, to make sure that that's true. You add DNA ligase to get bonding and a single vector gene of interest molecule. And then you take the mix. You usually have millions and millions of molecules that you're doing this with. And you take the whole mix. And you insert it into the host bacterium, OK? You insert-- and the correct term is transform. You insert or transform the whole mix into host bacteria. And these are the bacteria that that vector likes to grow in. And the last step in this, then, is that not all the bacteria are going to get one of these molecules, these recombinant DNA molecules inserted into them. In fact, most won't. It's only a tiny fraction of a percentage that are going to get a molecule of vector plus gene of interest inserted. So you want to get rid of all the bacteria that don't have your recombinant clone. And this is where this selectable marker we mentioned comes in. You select for bacteria that have got the vector gene of interest construct. And usually, the selection is with a particular type of chemical that is an antibiotic. And so you put an antibiotic resistance gene into the plasmid. And that allows the bacteria that have got the vector plus the gene of interest to grow in the presence of antibiotics. But don't worry about that for now. But you select for bacteria and the gene of interest. And then this allows the vector GOI to specifically grow in the bacteria. And you can then go ahead and isolate and use the DNA from this growth mix. I've drawn this for you in this next slide, where you can see a gene of interest is cut. The plasmid vector with its origin of replication is cut. The two DNAs are ligated. They've got compatible ends. And you can actually get three outcomes of this ligation. You might get your gene of interest just circularizing. That's not going to be very productive. Those DNAs will just disappear later in the process. You might get the vector only that ligates. That can be a bit of a problem. And there are ways you can get rid of those. And then you might get the correct construct, which is your gene of interest plus the vector. And that's what you want. You take that ligation mix, and transform bacteria with it, and grow, as we said, a lot of the recombinant clone. The selectable marker comes in because you can grow your transformants-- the vector or the vector plus gene of interest-- on, in this case, ampicillin, which is an antibiotic. Normally, ampicillin will stop bacteria from growing. That's why, if you get a bacterial infection, antibiotics are the things that you take to treat it. You can use that. The bacteria-- we got antibiotics from bacteria. And now, we can use them in molecular cloning, OK? So here, there is an ampicillin resistance gene also on the plasmid vector. If you grow the bacteria in the presence of ampicillin, only the bacteria that took up the vector with ampicillin resistance will be able to grow, OK? So that's a way to get rid of all the other non-transformed bacterial clones. Good. You've got a lot of information now about how you put together vectors and inserts, or genes of interest. And part of it is the notion of compatible ends. So I want you to do some practice in this class exercise, and see if you are able to use the concepts in a practical way.