DNA cloning and recombinant DNA | Biomolecules | MCAT | Khan Academy

Let's talk a little bit about DNA cloning, which is all about making identical copies of a piece of DNA. Usually, it's a piece of DNA that codes for something we care about; it is a gene that will express itself as a protein that we think is useful in some way.

Now, you might have also heard the term cloning in terms of the Clone Wars and Star Wars, or Dolly the sheep, and that is a related idea. If you're cloning an animal or an organism like a sheep, well, then you are creating an animal that has the exact genetic material as the original animal. But when we talk about cloning and DNA cloning, we're talking about something a little bit simpler. Although, as we'll see, it's still quite fascinating. It’s identical copies of a piece of DNA.

So how do we do that? Well, let's say that this is a strand of DNA right over here, and I'm just drawing it as a line, but this is double-stranded. I'll just write it down: this is double-stranded. I don't want to have to take the trouble of keeping drawing the multiple strands. Actually, let me just try to draw the two strands just to remind ourselves. So there we go, this is the double-stranded DNA.

Let's say that this part of this DNA has a gene that we want to clone. We want to make copies of this right over here—gene to clone, gene to clone. Well, the first thing we want to do is we want to cut this gene out somehow. The way we do that is using restriction enzymes, and there are a bunch of different restriction enzymes.

I personally find it fascinating that we, as a civilization, have gotten to the point that we can find and identify these enzymes and we know at what points of DNA that they can cut. They recognize specific sequences, and then we can figure out which restriction enzyme we should use to cut out different pieces of DNA.

But we have gotten to that point as a systemization. So, we use restriction enzymes. We might use one restriction enzyme. Let’s use a different color here. That latches on right over here and identifies the genetic sequence right over here and cuts right in the right place. So that might be a restriction enzyme right over there.

Then you might use another restriction enzyme that identifies the sequence at the other side that we want to cut. So let me label those things; those are restriction enzymes—restriction enzymes.

So now, after you apply the restriction enzymes, you will have just that gene. You might have a little bit left over on either side, but essentially you have cut out the gene. You've used the restriction enzymes to cut out your gene.

Then what you want to do is you want to paste it into what we'll call a plasmid. A plasmid is a piece of genetic material that sits outside of chromosomes but that can reproduce along with the genetic machinery of the organism. Or it can even express itself just like the genes of the organism that are in the chromosomes express themselves.

So this is where we cut. Let me write this: we cut, we cut out the gene. Then we want to paste it into a plasmid. Plasmids tend to be circular DNA, so we will paste it into a plasmid.

In order for them to fit, there are often times these overhangs. So you might have an overhang over there; you might have an overhang over there. The plasmid that we're placing in might have complementary base pairs over the overhangs, which will make it easier for them to react with each other if they have these overhangs.

So let me— we're pasting it into the plasmid. This is amazing because, obviously, DNA isn’t stuff that we can manipulate with our hands the way that we would copy and paste things with tape. You're making these solutions, and you're applying the restriction enzymes.

The restriction enzymes are just in mass cutting these things; they're bumping in just the right way to cause this reaction to happen. Then, you're taking those genes and then putting them with the plasmids that happen to have the right sequences at their ends so that they match up.

Then, you also put in a bunch of DNA ligase—DNA ligase—to connect the backbones right over here. We also saw DNA ligase when we studied replication, so that is DNA ligase, which you can think of as helping to do the pasting.

Now we have this plasmid, and we want to insert it into an organism that can make the copies for us. An organism that's typically used, or a type of organism, is bacteria—E. coli, in particular. So, what we could do is let’s say that we have a vial right over here. You have a vial, and it has a solution in it with a bunch of E. coli— a bunch of E. coli.

You actually wouldn't be able to see it visually, but there's E. coli in that solution. Then, you would put your plasmids, which would be even harder to see in that solution. Somehow, we want the E. coli—we want the bacteria—to take up the plasmid.

The technique that's typically done is giving some type of a shock to the system that makes the bacteria take up the plasmids, and the typical shock is a heat shock. This isn't fully understood, how the heat shock works, but it does. People have been using this for some time.

If you have a bacteria, you have a bacteria right over here. It has its existing DNA—this is its existing genetic material right over there. Let me label this. This is the bacteria. You put it in the presence of our plasmids, and you apply the heat shock.

Some of that bacteria is going to take in the plasmid; it's going to take in the plasmid. Just like that, it’s going to take it in.

What you then do is you place the solution that has your bacteria—some of which will have taken up the plasmid—and you try to grow the bacteria on a plate. So let me draw that; so let me draw. Here we have a plate to grow our bacteria on, and it has nutrients right over here that bacteria can grow on. It has nutrients, it has nutrients.

You could say, "Okay, we'll put this here," and then a bunch of bacteria will just grow. You would see things like this, which would be many, many, many, many cells of bacteria. There would be colonies of bacteria. You could just let them grow.

But there's a problem here because I mentioned some of the bacteria will take up the plasmids and some won't. You won't know, "Hey, you know, when this bacteria keeps replicating, it might form one of these." It might form one of these colonies, so this is a colony that you like—so this one is a good colony; put a check mark there.

But maybe this colony is formed by an initial bacteria or a set of bacteria that did not take up the plasmid, so it won't contain the actual gene in question. So you don't want that one.

So how do you select for the bacteria that actually took up the plasmid? What you do is, besides the gene that you care about—that you want to make copies of—you also place a gene for antibiotic resistance in your plasmid.

Now you have a gene for antibiotic resistance here. Only the bacteria— I think it's amazing that we, as humanity, have are able to do these types of things. But now, only the bacteria that have taken up the plasmid will have that antibiotic resistance.

So what you do is, in your nutrients, you do nutrients plus antibiotics—plus an antibiotic. This one will survive because it has that resistance. It has that gene that allows it to not be susceptible to the antibiotics.

But these are not going to survive. They're not even going to grow because there's antibiotic mixed in with those nutrients. This is a pretty cool thing.

You started with the gene that you cared about, you cut and pasted it into our plasmid—let me write the labels down—into our plasmid that also contained a gene that gave antibiotic resistance to any bacteria that takes up the plasmid.

You put these plasmids in the presence of the bacteria. You provide some type of a shock, maybe a heat shock, so that some of the bacteria take it up. Then the bacteria start reproducing, and as it reproduces, it is also reproducing the plasmids.

Because it has this antibiotic resistance, it is going to grow on this nutrient-antibiotic mixture, and the other bacteria that did not take up the plasmids are not going to grow. Just like that, you can take this colony right over here and put it into another solution or continue to grow it, and you will have multiple copies of that gene that are inside of that bacteria.

Now, the next question—and I'm oversimplifying things fairly dramatically—is, well, how do you—you now have a bunch of bacteria that have a bunch of copies of that gene—how do you make use of it? Well, the bacteria themselves, let’s say that gene is for something you want to manufacture—say, insulin for diabetics.

Well, you could actually use that bacteria's machinery; we use its reproductive machinery to keep replicating the genetic information. But you can also use its productive machinery, I guess you could say. It's going to express its existing DNA, but it can also express the genes that are on the plasmid.

In fact, that's what gives the bacteria its antibiotic resistance. But it could, if this gene was, say, for insulin, well then the bacteria will produce a bunch of insulin— a bunch of insulin molecules—which you might be able to use in some way. I'm not going to go into all the details of how you get the insulin out and how you could make use of it.

But, needless to say, it's pretty cool that we could even get to this point.