Question

Write the difference between dNTPs and ddNTPs. What is the function of ddNTPs in Sanger’s method of DNA sequencing?

Hint:

ddNTPs lack the 3′-OH group, causing chain termination in Sanger sequencing, enabling determination of DNA sequence by fragment size.

Answer 1

Feature	dNTPs (Deoxynucleotide triphosphates)	ddNTPs (Dideoxynucleotide triphosphates)
Structure	Contain a 3′-OH group on the sugar (deoxyribose)	Lack the 3′-OH group on the sugar (dideoxyribose)
Role in DNA synthesis	Incorporated by DNA polymerase to elongate DNA chain	Incorporated to terminate DNA chain elongation
Effect on chain elongation	Allow addition of next nucleotide	Prevent addition of next nucleotide, causing chain termination
Presence in Sanger sequencing	Used as normal substrates	Used as chain terminators

Function of ddNTPs in Sanger’s method: ddNTPs act as chain terminators during DNA synthesis because the absence of the 3′-OH group prevents the formation of a phosphodiester bond with the next nucleotide. When a ddNTP is incorporated, DNA polymerase can no longer add nucleotides, resulting in termination of DNA strand elongation at specific points. This generates DNA fragments of varying lengths that can be separated by gel electrophoresis to determine the DNA sequence.