The dideoxynucleotides are known as the chain terminator nucleotides that abort the nucleotide chain due to lack of 3’ OH group.
Within the past 20 to 25 years the DNA sequencing technology has evolved massively in which the Sanger sequencing technique is one of the most powerful discoveries so far.
In 1977, Sanger postulated the method to sequence the DNA known as the chain termination method of DNA sequencing using the special type of nucleotides known as the ddNTP or dideoxynucleotides.
In DNA sequencing, the sequence of DNA or the order in which each nucleotide is located on a gene can be determined precisely.
Due to the limitations of the traditional PCR technique that can only identify alterations (some) but can’t give sequence information, the DNA sequencing techniques are originated. You can read our meaty article on the present topic: DNA Sequencing: History, Steps, Methods, Applications and Limitations.
With Sanger sequencing, other sequencing techniques such as the Maximum-gilbert sequencing and various other semi-automated techniques were also evolved. Though the sanger sequencing platform was so powerful and handy on those days.
The entire technique of sanger sequencing relies on the use of ddNTPs. ddATP, ddGTP, ddCTP and ddGTP when incorporated into the amplification reaction, the chain got terminated.
This means the synthesis reaction can’t progress. In the present article, I will explain to you the role of ddNTPs in the sequencing methods. But before that let us understand the technique Sanger sequencing, broadly.
ddNTPs and Sanger sequencing:
The dideoxynucleotides or the ddNTPs are the artificially synthesized special type of nucleotides used in the Sanger sequencing technique.
The ddNTPs are different from the normal dNTPs as they don’t have the hydroxyl group at the 3’ position of the nucleotide.
Instead, both the 2’ and 3’ end of the ddNTP nucleotides have the hydrogen group in it. The structure of all dNTP and ddNTP is explained here:
Role of ddNTPs in Sanger sequencing:
Simply put, Sanger sequencing is a combination of amplification and polyacrylamide gel electrophoresis.
Taq DNA polymerase, normal dNTPs, template DNA and DNA primer are the ingredients of the sanger technique, much like the conventional PCR, but here a special extra ingredient is also employed.
Four different kinds of labeled ddATP, ddGTP, ddCTP and ddTTP are employed in the amplification. All fours are labeled with different color dye.
During the synthesis of DNA, the Taq DNA polymerase incorporates nucleotides to the growing DNA strand. First, it recognizes the 3’ end of the primer and then starts the synthesis reaction.
Meanwhile, it forms the phosphodiester bond to the adjacent nucleotide using the 5’ P group of the nucleotidetides. But when the ddNTP is incorporated, Taq DNA polymerase stops working!
The reason is that it can’t get the free 3’ end to elongate the chain, the ddNTPs have two hydrogen groups but not hydroxyl! Hence the polynucleotide chain progression stops.
Likewise, every time when the polymerase encounters the ddNTP nucleotide, it stops synthesis.
The end of the ddNTP-dideoxynucleotides are labeled with the color dye, the color signal is recorded by the computer.
Or different fragments of DNA appear when we run it on the polyacrylamide gel electrophoresis.
Related article: Preparing “DNA for Sequencing”.
dNTPs vs ddNTPs
As we said, though both are the type of nucleotides, dNTPs and ddNTPs are different from each other.
The dNTPs structurally have one hydroxyl group at the 3’ position of the sugar of nucleotide. Contrary, the ddNTPs don’t have OH or hydroxyl groups on the 3’ end.
The dNTPs have a hydrogen group at the second position of the sugar while the ddNTPs have a hydrogen group at 3′ and 2’ position of the sugar.
The 3’ OH end of the deoxynucleotides facilitate DNA synthesis by Taq DNA polymerase while due to the lack of the 3’ end, the Taq DNA polymerase can’t elongate the DNA chain when it encounters the ddNTPs.
The dNTPs are used in DNA sequencing and Polymerase chain reaction whilst the ddNTPs are only used in the Sanger sequencing technique to sequence DNA.
The traditional sanger sequencing technique was so time-consuming, interpreting each band of DNA in a PAGE needs high-end expertise and precision.
Nowadays, a robust and semi-automated version of the same technique is available, though it uses the same ddNTPs mediated-chain termination method, the signal is given by the termination is recorded by the computer and generates the peak of various nucleotides as per the fluorescence emitted.
On the downside, the Sanger sequencing can only sequence 1000 to 1500 bases precisely.