What is the principle behind Sanger sequencing, and what is its typical read length?

• A. Real-time monitoring of nucleotide incorporation during DNA synthesis.
• B. Chain-termination sequencing using dideoxynucleotides; typical read length up to ~800-1000 bases.
• C. Parallel sequencing of millions of fragments.
• D. Sequencing by measuring DNA mass changes.

Answer: (B)

Sanger sequencing relies on chain termination using dideoxynucleotides. In the reaction, DNA polymerase copies the template but, along with normal nucleotides, a small amount of ddNTPs is included. When a ddNTP is incorporated, the absence of the 3' hydroxyl prevents the next phosphodiester bond from forming, so the strand stops at that base. This creates a population of DNA fragments that all end at every possible base. By separating these fragments by size (traditionally on a gel, now often via capillary electrophoresis) and detecting which base terminates each fragment, you can read the sequence from the shortest to the longest fragment. This method is known for producing highly accurate reads of individual fragments.

The typical read length for Sanger sequencing is up to about 800 to 1000 bases per read, with practical reads often in the 700–900 base range. This contrasts with real-time monitoring of nucleotide incorporation during synthesis, which describes real-time sequencing-by-synthesis approaches, and with methods that sequence millions of fragments in parallel or that detect sequencing by measuring DNA mass changes.