So the ability to clone genes is the basis for all modern genetic advancements. I mean, the majority of things that we know about genetics today is due to the fact that we can clone genes and actually be able to study them one by one. So I'm going to talk to you about what genetic cloning is and the steps to do it. Essentially, genetic cloning is just taking some kind of DNA of interest and putting it into a system where you can develop a lot of it to study. Genetic cloning starts with amplifying the DNA of interest. You have some gene that you want a lot of copies of, and so you amplify it. There are many different ways to do this, but one, the most common, and the one mentioned in your book is polymerase chain reaction.
So a polymerase chain reaction consists of 3 steps. The first, is that you heat the DNA strands to a very high temperature, and that causes the double helix to separate into 2 single strands. Then, you lower the temperature and that allows for small, nucleotide primers to anneal, and that tells the polymerase where it's going to start. And in the third step, the polymerase comes on, binds to those primers, says, here I'm going to start, and that replicates the DNA. You do this in multiple cycles over and over again to create multiple copies of this DNA of interest. Now, you can do this using genomic DNA, but you can also use it to create and amplify cDNA from RNA. You take the RNA, you reverse transcribe it into cDNA, and then that DNA can also be used to amplify through PCR. Here is generally what PCR starts with. You have your first copy through cycle 1, you get 2. Through cycle 2, you get 4. And then each one of these creates 2, and it keeps going for as many cycles as you want. Typically, PCR does 20 to 35 cycles. So, you can get millions and millions of copies of DNA after these cycles. Like I said, if you start with DNA, this is a double strand because DNA is double-stranded. In the first step, you heat, and that creates 2 single strands that are now separated. Right? They're not bound. Then, in the second step, we move away, and may change the color too. Primers come in and anneal, say, here and here. And in the third step, the polymerase binds to the primer and amplifies. So then, what you end up with is 2 strands like this. And then you repeat this process over and over again, say 35 cycles, and you get a ton of DNA at the end.
So that's the first step. The second step to cloning is you take this DNA that you've amplified, and you have to cut it into small fragments. The way to do that is through restriction enzymes. Restriction enzymes are proteins; they chop DNA at very specific sequences. They create 2 types of ends: blunt ends and sticky ends. And these sticky ends are shown here. So if we have a DNA of interest, we take a restriction enzyme, which is here EcoR1. It cuts right here, this very specific sequence, it cuts at that sequence. And so you can see when it cuts, it creates 2 fragments, and these are sticky ends because their sequence overhangs. So these 4 nucleotides, they have nothing to bind to, and these 4 nucleotides also have nothing to bind to. And that's called a sticky end. If it was if these weren't here, and it was just this and this, those would be blunt ends. Now sticky ends the creation of sticky ends is super important, and typically the way that cloning happens. So if a blunt end occurs through that enzyme, it just happens to cut blunt ends. Usually, there has to be some kind of next step to create sticky ends because we're going to see the sticky ends are going to be very important. So the next step is pasting the DNA into a vector or a word that you might be familiar with is a plasmid, but essentially they're the same. And we call this whenever we take that DNA that we've amplified and now cut and we put it into a vector, we call that recombinant DNA.
So how you do this is you have DNA, it has a sticky end, so it has some nucleotides here that are free to bind, but they have nothing to bind to. And you use DNA ligase to bind those into a plasmid. So sticky ends, we usually make the sticky ends in the DNA itself but also in the plasmids. So you cut that plasmid with the same restriction enzyme that creates the same sticky ends. And that way, you can just paste that DNA straight into that plasmid. So like I said before, a vector use that term, but that's just a bacterial plasmid where the sequence of interest is placed. There are many different types of vectors with different characteristics for different organisms. If you have really large, I mean, like, kilobase upon kilobase fragments, DNA fragments, especially if looking at genomic DNA, these are called bacterial artificial chromosomes or yeast artificial chromosomes. And these are the plasmids used for large inserts. And then for plant cells, a common one that's mentioned in your book is the T1 plasmid. So essentially, how this is done is you start out with DNA, and a restriction enzyme sort of cuts here and here. So now you have this with some sticky ends. Then you cut the same restriction enzyme with the vector, and that gets pasted right into the vector. And then you can put the vector into bacteria, which is the next step here, is that the recombinant DNA is placed into bacteria. There are a few different ways to do this, some involving heat, some involving electricity, some involving chemicals, but essentially there are ways to get this DNA into bacteria. You can also put it into a virus, but typically it's done in bacteria, and that creates this transgenic bacteria that now has a gene that's not originally a bacterial gene. So a transgene is a new gene introduced into an organism, so for this case, it's that red gene. And so now this bacterium is a transgenic organism because it contains a transgene. And so we do this. Why do we do this? Do we need to put this random gene into bacteria? Well, bacteria grow rapidly. Right? So each time this bacterium divides, which can be just essentially every few minutes, it's going to create a copy of this sequence. And it's going to grow it, and grow it, grow it. It's also going to create that protein. So this is going to get expressed, and it's going to create this, protein version. And so, we can isolate a ton of this vector, or we can isolate the protein and use each one of these to study, either the gene itself or the protein or any sort of the intermediates in between. And so this is why cloning is important because it allows us to create this protein to study or to use for medicine or whatever we want to do with it. So that is genetic cloning. Now, let's move on.
