Determining the DNA Sequence from a Gel

by Jason Amores Sumpter
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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.