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Because remember, the DNA polymerase will only initiate replication if the primer is present. | Sanger Sequencing of DNA .txt |
And what that means is we have to know what this initial sequence is on that DNA molecule. | Sanger Sequencing of DNA .txt |
Because to build the DNA primer, we have to know what the complementary sequence is to this group here. | Sanger Sequencing of DNA .txt |
So if this is ACG, then we have to build a primer that contains a sequence TGC. | Sanger Sequencing of DNA .txt |
And so we can build that in a laboratory. | Sanger Sequencing of DNA .txt |
And we also radioactively label that DNA primer because that will basically allow us to pinpoint exactly where that molecule is when we undergo the process of gel electrophoresis. | Sanger Sequencing of DNA .txt |
So we add a label DNA primer that is complementary to the three end of that single stranded DNA molecule that we want to sequence. | Sanger Sequencing of DNA .txt |
So this is the three end of that DNA molecule. | Sanger Sequencing of DNA .txt |
And remember, DNA polymerase reads from three to five and it builds from five to three. | Sanger Sequencing of DNA .txt |
And that's why this is the end that we actually want to build the DNA primer for. | Sanger Sequencing of DNA .txt |
So we add the DNA polymerase and we also add the four types of deoxy nucleus diet triphosphate. | Sanger Sequencing of DNA .txt |
So we add dATP dGTP dCTP and TTP. | Sanger Sequencing of DNA .txt |
And finally, the important component in the standard died oxy method is a tiny amount of one of the four types of Ddntp molecules. | Sanger Sequencing of DNA .txt |
Remember, we have four different types that can exist and that's because we have four different types of bases. | Sanger Sequencing of DNA .txt |
So this base could be Adamine, it could be Guanine, it could be Cytosine, or it could be Thymine. | Sanger Sequencing of DNA .txt |
And so we have four different types of Ggntp molecules. | Sanger Sequencing of DNA .txt |
And in step two, we have to add a tiny amount, about 1% with respect to the other nucleoside triphosphates of a specific dig deoxy nucleuside triphosphate. | Sanger Sequencing of DNA .txt |
So we don't add all four types, we only add one type. | Sanger Sequencing of DNA .txt |
Now, why is that important? | Sanger Sequencing of DNA .txt |
Well, let's see what that actually does by examining the following diagram. | Sanger Sequencing of DNA .txt |
So we essentially take this DNA molecule, the single strand, and we mix it with these four components. | Sanger Sequencing of DNA .txt |
So we have the radioactively labeled DNA primer that is complementary to the three end. | Sanger Sequencing of DNA .txt |
We have the four types of deoxy nucleuside triphosphates. | Sanger Sequencing of DNA .txt |
These four ones shown here, we have the DNA polymerase, and we have a very small amount. | Sanger Sequencing of DNA .txt |
So let's say about 1% of the Ddatp. | Sanger Sequencing of DNA .txt |
So that's the specific Ddntp that we're going to choose for this specific experiment, for that specific beaker. | Sanger Sequencing of DNA .txt |
Now, what will begin to happen? | Sanger Sequencing of DNA .txt |
Well, what will happen is the complementary DNA primer will hybridize itself to this section here as shown in the following diagram. | Sanger Sequencing of DNA .txt |
So this is our DNA primer. | Sanger Sequencing of DNA .txt |
It will form these base pairs as shown in the following diagram. | Sanger Sequencing of DNA .txt |
So T base pairs with a G, base pairs with C, and C base pairs with G. And then the DNA polymerase will bind onto the primer and it will use the hydroxyl group on this cytosine to basically form that first phosphodiaester bond. | Sanger Sequencing of DNA .txt |
And so it will take a thymine. | Sanger Sequencing of DNA .txt |
It will take this molecule here to basically form the first base. | Sanger Sequencing of DNA .txt |
Then it will move on onto the second base, which is a T, and that will basically form an A. | Sanger Sequencing of DNA .txt |
Now, we have two types of A's that we can use. | Sanger Sequencing of DNA .txt |
One of them is the normal deoxy adenosine triphosphate. | Sanger Sequencing of DNA .txt |
The other one is a dioxy adenosine triphosphate. | Sanger Sequencing of DNA .txt |
And the dioxia adenosine triphosphate does not contain a hydroxyl group on the third prime carbon. | Sanger Sequencing of DNA .txt |
And what that means is if that DNA polymerase actually uses the Ddatp to place this base, it will not be able to continue that DNA replication process because that molecule does not have the hydroxyl group that is needed to produce the phosphor diester bond with the next base. | Sanger Sequencing of DNA .txt |
And so if this A comes from the Ddatp, this process will end and this will be the molecule that we will synthesize. | Sanger Sequencing of DNA .txt |
And this is why we have fragment number one, molecule number one. | Sanger Sequencing of DNA .txt |
Now, because we only have 1% of this Ddatp, this DNA polymerase will only sometimes use the Ddatp. | Sanger Sequencing of DNA .txt |
Usually it's going to use the normal dATP molecule. | Sanger Sequencing of DNA .txt |
And if it uses the normal dATP that contains the hydroxyl, then it will continue synthesizing those fossil diester bonds and so we can produce fragment number two. | Sanger Sequencing of DNA .txt |
So if this was normal, then it will continue. | Sanger Sequencing of DNA .txt |
So the DNA polymerase would add the thymine, then the guanine, then the cytosine, then the thiamine. | Sanger Sequencing of DNA .txt |
And now it comes to a T. So that means there is once again the possibility for an A. | Sanger Sequencing of DNA .txt |
And the A can either come from this normal dATP or the abnormal Ddatp that lacks the hydroxyl. | Sanger Sequencing of DNA .txt |
And if it's this group here, then once again we will stop the synthesis and this fragment will be produced. | Sanger Sequencing of DNA .txt |
Now, if it wasn't that molecule, then we would add the next. | Sanger Sequencing of DNA .txt |
So if the A was normal, the normal dATP, it would produce the next nucleotide in line. | Sanger Sequencing of DNA .txt |
And so the next one is also an A because this is a T. So once again, this is an A. | Sanger Sequencing of DNA .txt |
And now we have a possibility between this one or this one. | Sanger Sequencing of DNA .txt |
And let's suppose it's once again the ggatp. | Sanger Sequencing of DNA .txt |
And so it will stop it once again after this because it lacks that hydroxyl. | Sanger Sequencing of DNA .txt |
And so at the end in our mixture, in beaker number one, after we conduct step number two with the DD ATP, these are the other three fragments that will be present inside our beaker number one. | Sanger Sequencing of DNA .txt |
Now we take that beaker number one and we place it into SDS page. | Sanger Sequencing of DNA .txt |
So SDS polyacrylamide gel electrophoresis. | Sanger Sequencing of DNA .txt |
So this is our setup, and we have four different wells. | Sanger Sequencing of DNA .txt |
Now, well, number one, we label as the Ddatp, because this is step two, where we use the DD ATP. | Sanger Sequencing of DNA .txt |
We take the mixture and we place it into well number one, lane number one. | Sanger Sequencing of DNA .txt |
And we allow these to separate based on their masses. | Sanger Sequencing of DNA .txt |
Remember, in gel electrophoresis, the smaller our molecule is, the farther down it will move along that gel. | Sanger Sequencing of DNA .txt |
So if this is molecule one, Two and Three, this band will be for molecule One. | Sanger Sequencing of DNA .txt |
This band will be for molecule Two. | Sanger Sequencing of DNA .txt |
And this band, the highest up, will be the largest molecule, molecule Three. | Sanger Sequencing of DNA .txt |
Now, we continue the same process three more times. | Sanger Sequencing of DNA .txt |
And the second time around, we use a different Dgntp. | Sanger Sequencing of DNA .txt |
The third time around, we use yet another ggntp. | Sanger Sequencing of DNA .txt |
And the final fourth time, we use the final Dgntp. | Sanger Sequencing of DNA .txt |
So let's suppose the second time around, instead of using the DG ATP, we used Ddgtp. | Sanger Sequencing of DNA .txt |
And so instead of having the fragments where we stopped on the A's, we're going to have the fragments where we stop on the g's. | Sanger Sequencing of DNA .txt |
And so, because we only have two CS, so this C doesn't count because it's part of the primer. | Sanger Sequencing of DNA .txt |
So we have one C and we have a second C. So the complementary would have a g here and a g here. | Sanger Sequencing of DNA .txt |
So we only form two fragments. | Sanger Sequencing of DNA .txt |
And this lane would contain two bands because we only contain two fragments with different sizes. | Sanger Sequencing of DNA .txt |
Then we repeat the process. | Sanger Sequencing of DNA .txt |
Instead of using this one, we use DDCT. | Sanger Sequencing of DNA .txt |
So C, that means we have to count up the GS here, not including this one, because it's part of the primer. | Sanger Sequencing of DNA .txt |
So we have one g, we have two GS. | Sanger Sequencing of DNA .txt |
And that means we're going to have two complementary CS. | Sanger Sequencing of DNA .txt |
So we're going to have two different fragments once again, one, two. | Sanger Sequencing of DNA .txt |
And finally, if we use this one, we have to look for our adenine. | Sanger Sequencing of DNA .txt |
So we have to look for the adenine. | Sanger Sequencing of DNA .txt |
So we have one, two, three and four. | Sanger Sequencing of DNA .txt |
We should have four fragments. | Sanger Sequencing of DNA .txt |
And that's exactly what we get in this particular case. | Sanger Sequencing of DNA .txt |
So basically, in step three, we take step two and we repeat that same step three different times with the other three Ddntps. | Sanger Sequencing of DNA .txt |
And in step four, once the four reactions are completed, we run gel electrophoresis. | Sanger Sequencing of DNA .txt |
Each reaction mixture is placed into a lane. | Sanger Sequencing of DNA .txt |
So lane one, lane two, lane three, lane four. | Sanger Sequencing of DNA .txt |
And the results are then transferred onto a polymer sheet. | Sanger Sequencing of DNA .txt |
And then we use X ray order radiography to basically determine exactly where those radioactively labeled fragments actually were. | Sanger Sequencing of DNA .txt |
And so this is the diagram that we get. | Sanger Sequencing of DNA .txt |
Now, how can we use this to actually determine what the sequence of that initial DNA molecule is? | Sanger Sequencing of DNA .txt |
Well, we know what the first three nucleotides are because that's the primer. | Sanger Sequencing of DNA .txt |
So we have T, G and C. So the question is, what are these remaining nucleotides here? | Sanger Sequencing of DNA .txt |
Well, let's try to use the following setup to base determine what the sequence is. | Sanger Sequencing of DNA .txt |
So we know that the farther down along our page, the the smaller our fragment is and the closer the nucleotide sequence is to the beginning. | Sanger Sequencing of DNA .txt |
And the fragment all the way at the bottom basically describes this right over here. | Sanger Sequencing of DNA .txt |
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