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

Characteristic	dNTPs (Deoxynucleotide triphosphates)	ddNTPs (Dideoxynucleotide triphosphates)
Structure	Possess a 3′-OH group on the deoxyribose sugar.	Lack the 3′-OH group on the dideoxyribose sugar.
DNA Synthesis Role	DNA polymerase incorporates them to extend the DNA chain.	DNA polymerase incorporates them to halt DNA chain elongation.
Chain Elongation Effect	Permit the addition of subsequent nucleotides.	Inhibit the addition of subsequent nucleotides, leading to chain termination.
Sanger Sequencing Usage	Serve as standard substrates.	Function as chain terminators.

ddNTPs' role in Sanger sequencing: In Sanger's method, ddNTPs function as chain terminators. Their lack of a 3′-OH group prevents the formation of a phosphodiester bond with the next nucleotide, thereby stopping DNA polymerase activity and halting strand elongation at specific sites. This results in a collection of DNA fragments of varying lengths, which are then separated by gel electrophoresis to ascertain the DNA sequence.