Q.9 The nucleotide analogue used in DNA sequencing by chain termination method is
(A) 1′,3′-dideoxy nucleoside triphosphate
(B) 2′,3′-dideoxy nucleoside triphosphate
(C) 2′,4′-dideoxy nucleoside triphosphate
(D) 2′,5′-dideoxy nucleoside triphosphate
Sanger sequencing revolutionized genomics through chain-terminating nucleotide analogs that halt DNA synthesis at specific bases. This MCQ tests the precise chemical structure essential for biotech sequencing workflows and molecular biology exams.
Correct Answer
(B) 2′,3′-dideoxy nucleoside triphosphate is the chain terminator in Sanger’s method. ddNTPs lack hydroxyl groups at both 2′ and 3′ ribose positions, preventing phosphodiester bond formation after incorporation, generating fragments of defined length for sequence readout.
Option Analysis
(A) 1′,3′-dideoxy nucleoside triphosphate
1′,3′-ddNTPs don’t exist in standard biochemistry; the 1′ position anchors the nucleobase, not targeted for modification in chain termination chemistry.
(C) 2′,4′-dideoxy nucleoside triphosphate
No 4′-OH exists on ribose (deoxyribose lacks it naturally); 2′,4′-ddNTPs represent invalid chemistry irrelevant to DNA polymerase function.
(D) 2′,5′-dideoxy nucleoside triphosphate
5′ carries the triphosphate for polymerization; removing its OH disrupts all nucleotide incorporation, not selective termination.
Mechanism Essentials
DNA polymerase incorporates ddNTPs randomly against template bases in four parallel reactions (ddATP/ddCTP/ddGTP/ddTTP). Missing 3′-OH blocks 5′-phosphate linkage to incoming dNTPs, yielding ladder fragments separated by capillary electrophoresis for fluorescent base calling.
