Dideoxy Sequencing

In this video we're going to begin our lesson on Di di oxy sequencing. And so Di di oxy sequencing is going to be a specific DNA sequencing method or technique that uses D. D. N. T. P. S. Or die de oxy nucleotides as elongation terminators in order to help determine the sequence of the D. N. A. Now, di di oxy sequencing was actually first discovered way back in 1977 by a scientist named Frederick Sanger. And for that reason sometimes it is commonly referred to as just Sanger sequencing. And really this was the first method of DNA sequencing that uses these Di di oxy nucleotides. And so we'll be able to talk more about this Di di oxy sequencing and singer sequencing as we continue to move forward in our course. So I'll see you all in our next video.

in this video, we're going to focus on the specific components that are needed for diet the Oxy sequencing. But we're not going to get into the actual process of diet the oxy sequencing until we get to another video later on. In our course in this video, we're only focusing on the specific components, and so the components that are needed and died. The Oxy sequencing reactions include the following five components that we have labeled down below a through E. And, of course, these labels a through E that you see here correspond with the labels a through E that you see down below in our image. And so the very first component that is needed in a specific diet the Oxy sequencing reaction is, of course, going to be the unknown template D n a. Of interest, whose sequence we do not know, which is why we want to conduct di di oxy sequencing. And so over here, what we have is our template, d n A. And you can see this is the d n a. Here, and we do not know the sequence of the DNA, which is why we want to conduct this sequencing to figure out what is the sequence of this DNA. So that is the first component, then the second component that we are going to need is actually a d n a prelim arrays, which, you might recall, is an enzyme that prelim arises or builds d n A. And so DNA polymerase is the main enzyme that's needed for DNA replication. And so what you'll see here down below is the second component that we need is the DNA preliminaries. And, of course, this is the components of Di di Oxy sequencing. Okay, Now, uh, the third component that is needed is going to be DNA primers, and these DNA primers are going to a kneel to the template strand. And so recall that DNA primers were used in the specific technique that we talked about in our previous lesson videos called proliferates, Chain reaction, or PCR. And so what we'll see moving forward is that Di di Oxy sequencing is actually going to use a special type of PCR, and we'll see that moving forward now, the, uh, DNA primers you can see down below right here. Uh, and there will need to be, of course, uh, to DNA primers. Now, the the fourth component that is going to be needed are all for D n a, uh, deoxyribonucleic nucleotides. And so these are going to be the normal deoxyribonucleic tides that are used in, uh, d n A during normal cellular DNA replication. And so these deoxyribonucleic tides include D A t p d t t p d g t p and D c T p. So this is basically the Adnan timing guanine and cytosine, And then you can see these deoxyribonucleic tides down below. Here and again, these are the normal DNA nucleotides. And then, of course, for Di di oxy sequencing, where the fifth and final component that we're going to need is a small amount of a single die d oxy rebo nucleotide. And so this is the special type of nucleotide the D d a t p s d t t p S d d g t p s and D. D uh C T. P s. And so, in a particular deity oxy sequencing reaction within a single test tube, we would only want to use just one single, uh, nucleotide. And so we would have to separate. Uh, these reactions using different test tubes to use different dia de accion nucleotides. And we'll talk more about this as we move forward in our course. And so recall that these ddn tps these die deoxyribonucleic tides, they are going to terminate the DNA synthesis due to the presence of a three prime hydrogen atoms or groups. And so what you'll notice is down below right here. You can see that these are the die d oxy Revo, uh, DNA nucleotides. And so these are the special chain terminating uh, D d N T. P s diary, Oxy nucleotides. And so these are really the five major components that are needed for Diaby Oxy sequencing. And these are the components that you will see mentioned as we move forward and talk more about the specifics of the process of diet, the Oxy sequencing. And so, for now, this year concludes this lesson, and we'll be able to talk more about this as we move forward.

in this video, we're going to continue to talk about di di Oxy sequencing as we talk about the chain termination PCR steps or the chain termination proliferates, chain reaction steps. And so recall from our previous lesson videos that we already talked about preliminaries, chain reaction or PCR. And so be sure to go check out those older videos on PCR polymerase chain reaction before you continue here. Now. Also, recall from our previous lesson videos that DNA synthesis reaction is actually terminated when a di di oxy nucleotide or a D d NTP is added to the three prime men of the growing DNA strand. And really the use of these d d N t. P s to terminate the chain is really what chain termination PCR relies on. And so, in the first two steps of deity oxy sequencing, it requires setting up a chain termination p c R, which is really just a PCR reaction that's going to include small amounts of D. D N T. P s. And so what you can see here is that in this first step, we're going to need to set up four separate reactions in four separate test tubes And so notice down below in our image over here on the left hand side, you can see that we've got these four different test tubes where we're going to set up four different reactions and each of these four separate reactions that are being set up, they're each going to contain all of the components that are needed for a normal PCR. And they're also going to contain a small amount of a different dd NTP and really, this small amount of different DD NTP is what distinguishes one tube from another test tube. And so what you can see here is that over here in this, uh, test tube, it has all of the components for a normal PCR. But it also includes the d d NTP for cytosine. And so basically, this is going to provide chain termination at all of the side. I've seen, uh, nucleotides upon, uh, amplification of the d N A. Then, in this test tube noticed that it also has all of the normal components, or PCR, but it differs from the previous one and that it has the d d NTP for third timing and so chain termination in this tube is going to occur at all of the timing nucleotides. Then what we have over here in this test tube is again. It's going to contain all of the components for a normal PCR. But it's also going to contain a small amount of the D d N T p for a Dinneen A's. And so the same goes for this one over here, this last test tube. It's going to have all of the components for a normal PCR, but it differs in that it has a small amount of the DDM TPS for wanting. And so these four test tubes differ from each other in the small amount of the different DD NTP. That's being added. And again, the D D NTP is going to lead to chain termination at that specific nucleotide season. The first two teas in the second A's in the third and GS in the fourth. And so what you can see up at the top here is the mystery, D n A. And the mystery D N A. Is the specific DNA sequence that we try, uh, that we want to be able to sequence and determine the sequence of this mysterious D n a whose sequence we do not know at the moment. And so die. The Oxy sequencing can help us determine the sequence of this mystery D n A. And we have to set up chain termination PCR. And so this mystery d n A. Is going to serve as the template DNA for amplification during this PCR. And so the mystery DNA is going to go into all four of these test tubes. Um, Then in step number two, what we have is conducting the actual PCR reaction the chain termination PCR reaction. And so DNA synthesis, um, is actually going to produce a bunch of fragments of D N A. And the reason that it produces fragments of D N A is because of the d d N T P s, which is going to terminate the DNA synthesis reaction and create a bunch of fragments that terminate at the specific nucleotides that are indicated in each to. And so the DNA synthesis produces fragments of DNA, and these fragments of DNA are going to be complimentary to the unknown target. And again, the unknown target is the mystery DNA that we have here and so down below in our image. What you can see is that upon step number two conducting the actual chain termination PCR, the D N A is going to be replicated with chain termination P c R. And so what you see over here are the products. The PCR products and which will notice is that there are a bunch of different sized fragments of DNA that are going to be generated. Some of them only have one nucleotide, others are going to have more nucleotides. And what you can see is that we have all of these different sized fragments and the one the frag. For each of these fragments, you'll notice that in the background there is like a different colored background at the end of each of these chains and the one that has that different colored background here, Uh, that would represent the D. V M T. P. That's being, uh incorporated and terminating the chain at that nucleotide. And so what you can see here is you get all of these different sized PCR products and again these PCR products are going to be complementary to the mystery d n A. And so you can arrange these PCR products so that they are arranged based off of the size of the fragment. And so the size of the fragment is actually going to be revealing the nucleotides of the complementary PCR products from five prime 23 prime end. And so if we focus mainly on these di di oxy nucleotides that are at the end that terminate the chain, the PCR, um, these nucleotides here are going to be the ones that allow for generating these different sized fragments. And so, um, we can actually analyze these PCR products and analyzing these PCR products, um, is something that we're going to talk about in our next lesson video. And when these PCR products are analyzed, it can actually reveal the sequence of the mystery DNA. And so this year concludes our introduction to the chain termination PCR, and we'll be able to get some practice applying this and then talk about exactly how this, um, these PCR products can be analyzed to reveal the sequence of the DNA in our next video. So I'll see you all there

so after chain termination PCR The Next steps and died. The Oxy sequencing involved determining the d N a sequence from a gel. And so in the final two steps of didactic sequencing, the DNA sequence is finally going to be determined. And so in Step number three, which is a continuation of the chain termination PCR step from our previous lesson video the fragments from all four chain termination PCR reactions are going to be separated by size by using gel electro for racist. And so if we take a look at our image down below notice on the far left hand side, what we have are the products of our chain termination PCR, and these products are going to be different sized fragments. And so these different sized PCR products or different sized fragments they can be separated via gel, electro for Racists and recall from our previous lesson videos. That gel electrophoresis is going to load each of the different samples towards the top of the gel and specific wells, and then it will be separating the fragments within each lane based on their size. And so, in step number four, what we need to do is determined the sequence. And so the sequence of the DNA can be determined either manually using that gel from gel electrophoresis. Or that sequence can also be determined autonomously using a computer on a crime on what's known as a chroma to Graham, which is basically, uh, this plot that you see over here on the right. And so the way that we are going to focus on determining the sequence is going to be using the gel. And so the gel that you see down below right here, uh, it can actually be read backwards from bottom to top. And you read the gel across all lanes in order to reveal the complementary DNA sequence from five Prime 23 prime. And so I'll show you what I mean by this down below in this image. And so here, in the example of saying to determine the mystery DNA sequence by analyzing the gel electrophoresis results from dieting, oxy sequencing and so notice over here in this jail again, we have each of these lanes is going to contain, uh, a different, uh, chain termination PCR reaction from previous. And so that means that each of them is going to be ending with a different nucleotide. The ones with see here end with the nucleotide, see the ones with t ends with the nucleotide tea and so on the A's and with the A's and the G s are going to end with the G s. And so the shortest fragments are going to represent the fragments closest to the five prime end of the PCR product. And so that's why we want to reveal the sequence from five Prime 23 prime. Uh, then we need to start at the bottom. And so you read the gel backwards and notice that the band that is at the very bottom closest to the bottom is the one that is highlighted right here in Lane T. And so that means that this first nucleotide is going to be a T. And we can go ahead and put that here in this position as the first nuclear Italian, then reading the job backwards. The next one that's closest to the bottom is the yellow one. And so notice that we're looking across all lanes here, and the yellow one here is going to represent a G nucleotides, so that's going to be the next nucleotide G, Then the next one in the bottom here is an A. So we would put in a here. Then we have a C and another C So we get to back to back sees, then we have a t. Uh, and a and then last but not least, we have a G uh, in the final position towards the three prime end, and so you can see that the sequence has been revealed by reading this, uh, gel backwards from bottom to top, starting here these positions and working in this direction. And this is revealing from five prime 23 prime end. So then next, What we need to realize is, now that we've revealed the complementary DNA sequence, which is the sequence of the PCR products in order to reveal the mystery DNA sequence, we need to remember that the complementary DNA sequence is going to be complementary to the mystery DNA sequence. And so that means that we would need to just use our complementary base pairing rules to figure out the sequence of the mystery DNA and so recall that t s always base pair with a s on the opposite strands. So we have an A here GS always base pair with sees a s always base pair with teas, sees with G's C's with GS tees with a s A as with teas and G's with sees. And so what you see here is the mystery DNA sequence notice from three. Prime 25 Prime since recall that DNA sequences are, uh, DNA strains are gonna be anti parallel with respect to one another when they are complementary base parent. And so here we have revealed the sequence of the mystery D n A. And so you can see here how di the oxy sequencing and analyzing the gel backwards can be used to reveal the sequence. And so, once again, um, if the gel is not going to be analyzed manually, another way to analyze the DNA sequences by using a computer and the computer can generate a chroma to Graham, which is a plot that looks something like this. And the chroma to Graham is going to also reveal the sequence. And so that's a an alternative method of revealing the sequence. But this year concludes our brief lesson on how to determine the DNA sequence from the gel, uh, using diet, the oxy sequencing. And we'll be able to get some practice applying these concepts as we move forward in our course. So I'll see you all in our next video.