Sanger Dideoxy Sequencing Background Explanation:
DNA sequencing was originally developed in the 1970s. A variety of other methods were also developed around the same time but the chain termination method developed by Frederick Sanger rapidly became the method of choice.
DNA sequencing uses both normal nucleotides (A, T, G and C) but also chain terminating dideoxynucleotides (ddA, ddT, ddG, and ddC). When DNA polymerase incorporates a dideoxynucleotide of any base into a chain that is being extended, it causes the polymerase to stall and the chain can no longer be extended. This is pivotal for DNA sequencing reactions to work.
In order to sequence a piece of DNA the things that are required are:
- A primer that is specific to the region of DNA to be sequenced.
- A DNA template to be sequenced.
- Normal deoxynucleotides (A, T, G, and C)
- Dideoxynucleotides (ddA, ddT, ddG and ddC)
- DNA Polymerase
Just like when a normal cellular DNA polymerase extends DNA, during a sequencing reaction the growing DNA strand is extended in the 5’ to 3’ direction.
DNA sequencing reactions always contain more normal nucleotides than dideoxynucleotides. In the original method developed by Frederick Sanger, the sequencing reaction would be performed in four separate tubes, where each tube contained lots of normal nucleotides and a small amount of either dideoxy G, T, A or C. The reason for this will become clear below.
The sequencing method works in the following way. The primer binds to the DNA template in the region to be sequenced. The polymerase then starts to extend the primer in the 5’ to 3’ orientation. As the primer extends, it more frequently incorporates normal nucleotides because there are more of those in the reaction mixture. So if the primer sequence was AAAATTTTGGGGCCCC and then the next nucleotides in the DNA to be sequenced were ATCGAAA then the primer (plus the new nucleotides) would now have the sequence AAAATTTTGGGGCCCCATCTAAA. Then if we imagine that in this reaction we have small amount of dideoxy guanosine in it, and that the next base is a G, we can assume that most of the time the extending chain will incorporate a normal G nucleotide (because there are more of them in the reaction). But occasionally the chain will incorporate a dideoxynucleotide and the chain will terminate with the sequence AAAATTTTGGGGCCCCATCTAAAG. However, those extending chains that did not terminate at the first G will continue to the next one, where the same possibilities apply, the chain will either terminate or carry one. For example, AAAATTTTGGGGCCCCATCTAAAGTAAAACCCG may be the next terminated DNA fragment. So where ever you have a G in the DNA sequence, you will have some chains that have terminated and are of a specific length. Now also imagine you have the same reaction for the other nucleotides A, C and T. Where each tube contains a population of DNA fragments that terminate at each of the nucleotides for which there is dideoxynucleotide in the reaction.
Now we run those four reactions of an agarose gel that separates the DNA by size. In the case above, we would see that the G reaction would produce DNA fragments that are 24 base pairs (bp) long and 33bp long. Whereas if we did the same reaction with a C dideoxynucleotide in the reaction, we would have had chains that terminated at 19bp, 30bp, 31bp and 32 bp long. The reaction with dideoxy T would produce fragments of 18bp, 20bp and 25 bp and the A reaction would produce fragments of 17bp, 21bp, 22bp, 23bp, 26bp, 27bp, 28bp and 29bp. If these are all run on a gel in adjacent lanes, it then becomes possible to see how we can read the DNA sequence. See below:
If you read the letters up from the bottom, you will see that it tells you the DNA sequence of the reactions described above. However, this method was quite time-consuming and required four different reactions to get a single sequence. For this reason, sequencing reactions are now performed with dideoxynucleotides where each different nucleotide is labelled with a different fluorphore. So each terminated chain has a colour that is associated with the last base in its chain. Then if the reaction is run on a gel you can see how the sequence can be read in the same way as before. It is now common to run the sample through a capillary tube and a computer detects the colour of each DNA fragment as it runs out of the end of the capillary. Using this technique it is common to obtain DNA sequence reads in excess of 1000 base pairs.
In this diagram the dideoxynucleotides are labelled as: ddA = Green, ddT = Red, ddC = Blue, and ddG = Black.
Author: Dr Ryan Cawood