Much of the progress that has been made in molecular biology in recent years has been in large part due to the ability to sequence an organism's DNA. Although large-scale efforts of this sort, such as the Human Genome Project, have used more advanced technology, basic DNA sequencing capability arose out of the dideoxy sequencing method. This method is therefore foundational and is the subject of this video. To understand the dideoxy sequencing method, it is important to have a firm grasp of basic DNA structure. DNA stands for deoxyribonucleic acid.
It is assembled from individual nucleotides. A nucleotide has three distinct parts. The phosphate group, the sugar, in this case deoxyribose, and the nitrogenous base, which could be A, G, C, or T.
In this case, it's T, thymine. To link individual nucleotides, a bond must form between the phosphate group of one and the sugar group of another. More specifically, the hydroxyl groups from the phosphate and the sugar combine to form a molecule of water, which is produced as the bond is formed. This process is known as dehydration synthesis because water is the byproduct. Now remember that with DNA, the sugar is deoxyribose.
This name comes from the fact that one hydroxyl group has already lost its oxygen. If we replaced deoxyribose with dideoxyribose, both hydroxyl groups would be missing their oxygen. In this case, an adjacent nucleotide would be unable to bond. because dehydration synthesis cannot occur.
This sequence stopping power of a dideoxynucleotide is at the core of dideoxy sequencing. Building on this stopping power, we must remember that there are four distinct nitrogenous bases. Thymine, cytosine, adenine, and guanine. Therefore, as we attempt to build a DNA molecule, it is possible for us to employ dideoxynucleotides for only one type of nucleotide, either A, G, C, or T. Therefore, we could stop DNA sequence construction only at sites where that particular base appears.
We can use this stopping power to decode a DNA sequence. In our first experiment, Experiment T, we'll use regular deoxynucleotides for A, G, and C. However, for half of the T nucleotides, we'll use a dideoxy form, giving the possibility of stopping the DNA sequence assembly.
If we do this experiment enough times, we'll obtain each of the following varieties of sequences. It might be the case that even though half of the nucleotides are dideoxy, none of them ever get inserted and we get the full sequence anyway. Or, perhaps, we insert a dideoxy T here and cut the sequence short by one.
Or, maybe, we insert the dideoxy T1 earlier and cut the sequence still shorter. Finally, it's possible that the first nucleotide we add is dideoxy and the sequence stops at 1. Therefore each of these four possibilities could exist when we use dideoxy-T. Now let's do another experiment.
In this one, experiment C, we'll use regular deoxynucleotides for A, G, and T, but half of our C nucleotides will be dideoxy. We do the experiment a number of times. and observe the following forms. Again, no dideoxes included.
The full sequence is obtained. We might, however, insert a dideoxy C in the first C position. Because C is at the final position of the sequence, it's difficult to determine if there is in fact a C there. or if we've just gotten the full length result. We can't exactly know for sure.
Let's do the same experiment for G. In this case, again, it's possible that we get the full sequence, or it's possible that at our first instance of G, we insert a dideoxy and the sequence stops. Therefore, there are two options for our G experiment. Finally, we'll repeat the experiment for A.
considering the possibility that we get the full sequence, or that at our first instance of A, a dideoxynucleotide is inserted and the sequence stops. However, you may be wondering how we actually analyze DNA sequence length. We can't exactly use a ruler.
Instead, we will make the DNA fragments have a race through a thick gel. Because we can't count on DNA to run by itself, We use an electrical current to pull the DNA. This process is called gel electrophoresis.
Small DNA molecules will travel furthest through the gel, while large molecules will have a hard time making progress and will therefore stay closer to the starting point. We will put the DNA molecules in lanes according to which dideoxynucleotide we were using for a given experiment. For experiment T, we'll put all of those results in a lane labeled T. Now we'll let the gel run.
Using the knowledge that the smallest sequences would probably make the furthest progress, and by considering the results of our race, we can reconstruct the sequence of nucleotides used to build this DNA sequence. The group traveling furthest was the smallest, one nucleotide. Because it's in lane T, we can check back and put a T in position 1. We can use this same process of analysis to reconstruct the entire sequence.
Our next furthest DNA sequence comes in lane G, so we put a G into our sequence. A, T, and T. This gives us a sequence of TGATT. What about our last position, though? The position occupied in all the lanes?
Well, because it's possible to build the full sequence without ever inserting a dideoxy stopper nucleotide, this sequence will appear in each lane. Therefore, we can't determine which nucleotide is in the final position. However, if we really wanted to know this base, all we would have to do is examine a bit longer segment.
Now you have a basic understanding of the technique that was fundamental in the modernization of genetics, molecular biology, and the study of organisms'genomes.