 Now, let's see how we can use PCR technology to sequence DNA. I will remind you that genome project which was basically sequencing human genome. 2.7 billion US dollars were spent to sequencing human genome. That's how important it was. It took 13 years to sequence the human genome. Let's see how we can sequence a DNA segment using PCR technology. First of all, let me introduce you to the DNA nucleotides once again. We are familiar with ribonucleosides. We are familiar with the deoxyribonucleosides. Here I'm going to introduce you a new type. This is D-dioxy, di-doxyribonucleoside. As you know, the carbon atom number 3 has the 3 prime hydroxyl, which is required for extending the DNA, DNA chain. If the 3 prime hydroxyl is not present, the DNA new nucleotide cannot be added to the growing DNA fragment. So, we use the di-doxyribonucleosides in a PCR reaction for sequencing. Let's see how that is done. We make 4 different types of di-doxy nucleotides, one for each base present in the DNA. And special thing about these di-doxyribonucleosides is that they contain a glowing molecule which is specific to each base. For example, C here is red color, G has blue color, T has green color, A has yellow color. So, these di-doxyribonucleosides will always have a molecule attached to it which will emit yellow color, T thymine will have a molecule attached to it which will emit green color, and G blue color, C the red color. So, we run our PCR reaction. We have the template DNA. We want to know what is the sequence of this DNA strand. We have a primer, we have little bit information about this DNA fragment. We have to know the little sequence so we can design the primer. The rest of the sequence we need to determine. So, we add these components, the normal components for PCR reaction, the normal nucleotides, and the enzymes. And additionally we add these special di-doxyribonucleosides attached to these relevant glowing molecules in a very, very small quantity compared to the normal nucleotides. Now, when we run the reaction, these bases are incorporated in at random. Say, for example, we had our primer and it was being extended. Here the primer has annealed. This is the primer sequence. There's a C-prime hydroxyl. The next base here, whatever base that is added here would be the base that is complementary to this question mark. So, when the base is added, if it is a normal base, it is fine. But if it is one of those special bases that we talked about, di-doxyribonucleotide bases attached to a specific color, the reaction will stop right here. So, we will basically generate a band, DNA band, which is short. Let's just, for the sake of argument, say it was A. So, actually, let's just say it was a G that will make our life a little easier. Let me raise this. So, let's just say that it was A. So, we had an A that got incorporated and this A was a special nucleotide. It stopped the DNA chain from the primer from extending any further. And now what happened is that we complete this reaction. This procedure is going on at random. In the next cycle, perhaps this chain will grow all the way up till here and then the next nucleotide that gets added, say, for example, G, it will be the special nucleotide. So, we will generate PCR products of all the lengths from our starting point of the primer to the last nucleotide. All these bands will be formed with the terminal nucleotide being the special nucleotide. Now, when we run the products of this PCR reaction on a gel, the shortest one will come out first. So, in this case, it was a G that has come out of the gel that this fragment has moved the fastest on the gel, followed by another G, followed by a C, T, so on and so forth. So, this was the first nucleotide that was added on the primer because it is the shortest one. So, as we keep on running our gel, these DNA strands will start moving. This gel is a special type of gel. It is basically a column gel. It is a glass column. At one end of this glass column, we have a laser that is going to emit a special light that will make our special D-dioxy ribonucleosides glow and we have a detector or a camera here that can detect what color was emitted. So, the first color that was emitted was blue and we know where the blue color was attached. Blue color was attached to G. Then again, a blue color was detected. That was also G. Then a red, which was C. T was then the next was green, which was T. So, you see, eventually as we move along, we are determining the sequence of our DNA, which can have very significant consequences. We need to sequence DNA for various purposes. We need to detect certain type of disorders to find different type of changes in DNA. So, we can sequence DNA using PCR technology. Here, I feel pertinent to mention that now things have changed a little bit. As I mentioned at the start of this module, that it took 2.7 billion US dollars and 13 years to sequence completely the human genome. Now, the technologies have already been discovered, have already been invented, that can accomplish the same feat in about a day or so at about a thousand US dollars per human genome. This is the speed at which technology is moving and now, the day is not very far that we can sequence when we go to our pathologist for a test, we can also ask them to sequence our genome for us.