Which sequencing method uses chain-termination with dideoxynucleotides to determine sequence?

• A. Next-generation sequencing
• B. Sanger sequencing
• C. RT-PCR
• D. Southern blot

Answer: (B)

This method relies on chain termination by dideoxynucleotides to read DNA sequence. In Sanger sequencing, DNA synthesis uses normal nucleotides plus small amounts of dideoxynucleotides that lack the 3' hydroxyl group. When a ddNTP is incorporated, the chain can no longer be extended, so many copies terminate at every possible position. Since each ddNTP corresponds to a specific base, the resulting fragment ends reveal the identity of the terminal base. By separating these fragments by length and reading the terminating base from smallest to largest, you obtain the sequence. This approach is classic, highly accurate for short reads, and foundational for understanding sequencing while newer methods offer much higher throughput. The other techniques listed serve different purposes: next-generation sequencing uses parallel, different chemistry for massive data throughput; RT-PCR measures RNA by converting it to DNA and amplifying it; Southern blot detects specific DNA sequences via hybridization after restriction digestion.