Question

In Sanger's chain termination method, incorporation of ddNTP cause the growing DNA chains to terminate prematurely. Explain how.
(ii) Briefly write the steps of Sanger's chain termination method of DNA sequencing.

Hint:

Make sure to remember the roles of different enzymes in the DNA replication process, and the method of chain termination by ddNTPs.

Answer 1

(i) Chain Termination by ddNTPs: Dideoxynucleotides (ddNTPs) are analogs of deoxynucleotides (dNTPs) used in DNA synthesis. They lack a 3'-hydroxyl group (-OH), which is required for the formation of a phosphodiester bond with the incoming nucleotide. Therefore, if a ddNTP is incorporated into a DNA strand by the DNA polymerase, no further nucleotide can be added to the chain, leading to premature termination of the DNA synthesis. 

(ii) Steps of Sanger's chain termination method of DNA sequencing: 
Reaction Setup: The DNA to be sequenced is used as the template in four different tubes. Each tube also contains a DNA polymerase, a primer, deoxynucleotide triphosphates (dNTPs), and one of the four dideoxynucleotide triphosphates (ddATP, ddGTP, ddCTP, or ddTTP), at a low concentration. 
DNA Synthesis: DNA polymerase starts to extend the primer. If any of the ddNTPs are incorporated into the new strand, it leads to premature chain termination. 
Chain Termination: Synthesis proceeds until a ddNTP is incorporated at random positions of the growing strand. As ddNTPs lack the 3’OH group, extension can no longer proceed. This generates many different chains of varying lengths, with the ddNTP at the terminating position.
 Gel Electrophoresis: The DNA fragments are then separated based on their size using gel electrophoresis. Here, smaller fragments move further down the gel, and larger fragments move slowly. 
Autoradiography/Visualization: The fragments are visualised in the gel using radioactive markers or fluorescent dyes. This produces a ladder-like image with the smallest band appearing at the bottom, and the sequence is read from bottom to top.