So after chain termination PCR, the next steps in dideoxy sequencing involve determining the DNA sequence from a gel. And so, in the final two steps of dideoxy sequencing, the DNA sequence is finally going to be determined. In step number 3, 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 using gel electrophoresis. If we take a look at our image below, notice on the far left-hand side what we have are the products of our chain termination PCR. These products are going to be different-sized fragments. These different-sized PCR products or different-sized fragments can be separated via gel electrophoresis. Recall from our previous lesson videos that gel electrophoresis loads each of the different samples toward the top of the gel in specific wells and then separates the fragments within each lane based on their size.
In step number 4, what we need to do is determine the sequence. The sequence of the DNA can be determined either manually, using the gel from gel electrophoresis, or the sequence can also be determined automatically using a computer on what is known as a chromatogram, which is this plot that you see over here on the right. We are going to focus on determining the sequence using the gel. The gel that you see below can actually be read backwards from bottom to top, and you read the gel across all lanes to reveal the complementary DNA sequence from 5' to 3'. I'll show you what I mean by this in the image below. In the example, it says to determine the mystery DNA sequence by analyzing the gel electrophoresis results from dideoxy sequencing. Notice over here in this gel, again, we have each of these lanes containing a different chain termination PCR reaction from the previous. That means that each of them is going to be ending with a different nucleotide. The ones with 'C' here end with the nucleotide 'C,' the ones with 'T' end with the nucleotide 'T,' and so on. The 'A's end with 'A's, and the 'G's are going to end with 'G's. The shortest fragments represent the fragments closest to the 5' end of the PCR product. To reveal the sequence from 5' to 3', we need to start at the bottom. You read the gel backwards and notice that the band at the very bottom, closest to the bottom, is highlighted here in lane 'T.' 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 nucleotide. Then reading the gel backwards, the next one closest to the bottom is the yellow one, representing a 'G' nucleotide, which is going to be the next nucleotide. Then the next one at the bottom here is an 'A,' so we would put an 'A' here. Then we have a 'C' and another 'C,' so we get two back-to-back 'C's.' Then, we have a 'T,' an 'A,' and last but not least, we have a 'G' in the final position toward the 3' end. You can see that the sequence has been revealed by reading the gel backwards, from bottom to top starting at these positions and working in this direction, revealing it from 5' to 3' end.
Next, we need to realize that now that we've revealed the complementary DNA sequence, which is the sequence of the PCR products, 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. So, we would use our complementary base pairing rules to figure out the sequence of the mystery DNA. 'T's always base pair with 'A's on the opposite strand, so we have an 'A' here. 'G' is always base paired with 'C's, 'A's always base pair with 'T's, 'C's with 'G's, 'C's with 'G's, 'T's with 'A's, 'A's with 'T's, and 'G's with 'C's. What you see here is the mystery DNA sequence from 3' to 5' since recall that DNA strands are gonna be anti-parallel with respect to one another when they are complementary base pairing. Here we've revealed the sequence of the mystery DNA. You can see how dideoxysequencing and analyzing the gel backwards can be used to reveal the sequence.
Once again, if the gel is not going to be analyzed manually, another way to analyze the DNA sequence is using a computer, which can generate a chromatogram, a plot that looks something like this, and the chromatogram is also going to reveal the sequence. This concludes our brief lesson on how to determine the DNA sequence from the gel, using dideoxy sequencing. We'll be able to get some practice applying these concepts as we move forward in our course. I'll see you all in our next video.