In this video, we're going to continue to talk about dideoxy sequencing as we discuss the chain termination PCR steps or the chain termination polymerase chain reaction steps. Recall from our previous lesson videos that we already discussed polymerase chain reaction or PCR. Be sure to check out those older videos on PCR, polymerase chain reaction, before you continue here. Also recall from our previous lesson videos that DNA synthesis reaction is actually terminated when a dideoxynucleotide or a ddNTP is added to the 3' end of the growing DNA strand. The use of these ddNTPs to terminate the chain is really what chain termination PCR relies on.

In the first two steps of dideoxy sequencing, it requires setting up a chain termination PCR which is just a PCR reaction that's going to include small amounts of ddNTPs. In this first step, we're going to need to set up 4 separate reactions in 4 separate test tubes. Notice in our image on the left-hand side, you can see that we've got these 4 different test tubes where we're setting up 4 different reactions. Each of these separate reactions will contain all of the components needed for a normal PCR and also a small amount of a different ddNTP, which distinguishes one tube from another. In one test tube, it has all of the components for a normal PCR, but it also includes the ddNTP for cytosine, providing chain termination at all of the cytosine nucleotides upon amplification of the DNA. In another test tube, it has all of the normal components but includes the ddNTP for thymine, so chain termination in this tube will occur at all of the thymine nucleotides. Another test tube will contain all of the components for a normal PCR and a small amount of the ddNTP for adenine, and similarly, the last test tube will have all the components for a normal PCR but with the ddNTP for guanine. These four test tubes differ from each other in the small amount of the different ddNTP added, leading to chain termination at specific nucleotides: C's in the first, T's in the second, A's in the third, and G's in the fourth.

At the top here is mystery DNA, the specific DNA sequence that we want to sequence and determine. Dideoxy sequencing can help us determine the sequence of this mystery DNA, and we have to set up chain termination PCR for this. The mystery DNA will serve as the template DNA for amplification during this PCR and will go into all 4 of these test tubes. Then, in step number 2, we conduct the actual PCR reaction, the chain termination PCR reaction. DNA synthesis will produce a bunch of fragments of DNA because of the ddNTPs which will terminate the DNA synthesis reaction and create fragments that terminate at the specific nucleotides indicated in each tube.

The DNA synthesis produces fragments of DNA that will be complementary to the unknown target, the mystery DNA. Below in our image, upon conducting the actual chain termination PCR in step number 2, the DNA is replicated. You can see the products, the PCR products, and notice that there are various sized fragments of DNA generated. Some fragments have only one nucleotide, others more, and there's a different colored background at the end of each chain that represents the ddNTP being incorporated and terminating the chain at that nucleotide. By arranging these PCR products based off the size of the fragment from 5' to 3' end, the dideoxynucleotides at the chain's end allow for generating these different-sized fragments. Analyzing these PCR products is something we're going to talk about in our next lesson video. Analysis of these PCR products can reveal the sequence of the mystery DNA. This concludes our introduction to the chain termination PCR, and we'll be able to get some practice applying this and discuss exactly how these PCR products can be analyzed to reveal the sequence of the DNA in our next video. See you all there.