Describe the Sanger chain‑termination method of DNA sequencing and explain how the incorporation of dideoxynucleotides allows the sequence to be read.
Answer
In Sanger sequencing, DNA polymerase extends a primer in four separate reactions, each containing a normal dNTP and a small amount of one specific ddNTP; incorporation of a ddNTP terminates elongation, producing fragments of varying lengths that end at each occurrence of that base, and separating the four sets on a gel reveals the DNA sequence.
Step-by-step solution
- State that the method uses four separate reaction tubes, each supplied with all four dNTPs plus a low concentration of one type of ddNTP (ddATP, ddTTP, ddGTP or ddCTP).
- Explain that DNA polymerase adds nucleotides to a primer annealed to the template strand.
- Highlight that when a ddNTP is incorporated, it lacks a 3′‑OH group, so further extension cannot occur – the chain is terminated at that point.
- Because the ddNTP concentration is limiting, termination occurs at random positions, generating a collection of fragments ending at each occurrence of the specific base.
- After the reactions, the fragments are separated by size using electrophoresis; the smallest fragments travel farthest.
- By reading the order of bands from the bottom (shortest) to the top (longest) across the four lanes, the original DNA sequence is deduced base by base.
Common mistake: Believing ddNTPs are incorporated continuously like normal dNTPs, thus not realizing they cause chain termination
Concept tested: Sanger DNA sequencing principle
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