What is the primary principle behind Sanger sequencing? Select all correct answers.

Question

BlackTom AI · Accepted Answer

Sanger sequencing relies on chain termination by dideoxynucleotides to produce fragments of varying lengths. Mechanism and workflow: a single-stranded DNA template is primed and a DNA polymerase extends the primer in a mix containing normal deoxynucleotides (dNTPs) plus a small proportion of chain-terminating dideoxynucleotides (ddNTPs). Each ddNTP lacks the 3′-OH group required to form the next phosphodiester bond; when a ddNTP is incorporated, extension stops at that position. Because ddNTPs for A, T, C, and G are each labeled with a distinct fluorescent dye (dye-terminator method), synthesis produces a population of terminated fragments of all lengths ending at every occurrence of each base. These fragments are separated by size using capillary electrophoresis; as they pass a detector, the dye color identifies the terminal base for each fragment. Base-calling software converts the fluorescence trace into a sequence and writes the results into an output file.

An .abI file is generated, which contains the sequence of the PCR product. Modern Applied Biosystems (ABI) Sanger instruments produce a chromatogram file (commonly seen as .ab1/.abi/.abI formats) that stores the raw fluorescence trace, quality metrics, and the base calls (the called sequence) derived from the electropherogram. When a PCR product or cloned insert is used as template, the called sequence in the .abI file corresponds to that PCR product.

Why the two statements are correct: the first statement describes the fundamental chemical basis of Sanger sequencing (ddNTP-mediated chain termination); the second describes the practical output of the sequencing run (an ABI chromatogram file containing the called sequence). Common incorrect statements would attribute the method to sequencing-by-synthesis with reversible terminators (Illumina), sequencing-by-ligation (SOLiD), or single-molecule real-time detection (PacBio/nanopore)—those are different technologies. Therefore, the two provided answers are correct and match the authoritative answer.

What is the primary principle behind Sanger sequencing? Select all correct answers.多项选择题

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