Q.23 Which one of the following is the basic principle of Sanger’s DNA sequencing
method?
(A) Chain termination by incorporation of dideoxynucleotides
(B) Chain elongation by incorporation of dideoxynucleotides
(C) Release of inorganic pyrophosphate
(D) Chain cleavage by modification of dideoxynucleotides

Sanger’s DNA sequencing, also known as the chain-termination method, determines the order of nucleotides in DNA by selectively stopping chain extension. The basic principle relies on dideoxynucleotides (ddNTPs) that terminate DNA synthesis upon incorporation.​

Correct Answer

(A) Chain termination by incorporation of dideoxynucleotides.​

This option accurately describes the core mechanism: DNA polymerase incorporates normal dNTPs to extend the chain but stops when a ddNTP (lacking a 3′-OH group) is added, preventing phosphodiester bond formation. Fragments of varying lengths are generated and separated by electrophoresis to read the sequence.​

Option Analysis

(B) Chain Elongation by Incorporation of Dideoxynucleotides

ddNTPs do not promote elongation; they halt it by missing the 3′-OH group needed for further nucleotide addition. This misrepresents the method, as chain growth relies on regular dNTPs.​

(C) Release of Inorganic Pyrophosphate

Pyrophosphate release occurs during normal nucleotide incorporation in both Sanger and other methods like pyrosequencing, but it is not Sanger’s distinguishing principle. Sanger specifically uses chain termination, not pyrophosphate detection.​

(D) Chain Cleavage by Modification of Dideoxynucleotides

Sanger sequencing involves no chemical cleavage; termination happens via enzymatic incorporation of ddNTPs during synthesis. Cleavage methods apply to techniques like Maxam-Gilbert sequencing.​