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 deoxynucleotide (dNTP) analogs used in DNA synthesis. They lack the 3'-hydroxyl group (-OH) necessary for phosphodiester bond formation with incoming nucleotides. Consequently, DNA polymerase incorporation of a ddNTP halts DNA chain elongation, resulting in premature termination of DNA synthesis.

(ii) Steps of Sanger's Chain Termination DNA Sequencing Method: 
Reaction Setup: The DNA target serves as the template in four distinct tubes. Each tube contains DNA polymerase, a primer, deoxynucleotide triphosphates (dNTPs), and a low concentration of one of the four dideoxynucleotide triphosphates (ddATP, ddGTP, ddCTP, or ddTTP). 
DNA Synthesis: DNA polymerase initiates primer extension. Premature chain termination occurs upon incorporation of any of the ddNTPs into the nascent strand. 
Chain Termination: Synthesis continues until a ddNTP is randomly incorporated into the growing strand. Due to the absence of the 3’OH group, further extension is blocked. This process generates numerous DNA fragments of varying lengths, each terminating with a ddNTP.
 Gel Electrophoresis: The DNA fragments are subsequently separated by size via gel electrophoresis. Shorter fragments migrate further down the gel, while larger fragments move more slowly. 
Autoradiography/Visualization: Fragments are visualized within the gel using radioactive labels or fluorescent dyes. This yields a ladder-like pattern, with the smallest fragment at the bottom, allowing the sequence to be read from bottom to top.