Sanger sequencing utilizes chain-terminating dideoxynucleotides. Which statement correctly describes its limitation?

Sanger sequencing utilizes chain-terminating dideoxynucleotides. Which statement correctly describes its limitation?

• A. It uses chain-terminating dideoxynucleotides with labeled fragments; limited read length and throughput
• B. It relies on massively parallel sequencing for all fragments
• C. It sequences RNA directly
• D. It cannot determine sequence at all

Answer: (D)

Sanger sequencing uses chain-terminating dideoxynucleotides to create a set of DNA fragments that end at each possible base. When a ddNTP is incorporated, DNA polymerization stops because the molecule lacks the 3' OH needed to continue growth. By labeling these terminated fragments and separating them by size, you read off the sequence from the shortest to the longest fragment. The main limitation is read length and throughput. Because you generate and read one fragment at a time per reaction lane, the readable stretch per run is relatively short (a few hundred bases in typical setups), and processing many fragments becomes time-consuming and labor-intensive. This makes Sanger sequencing low-throughput compared with newer, massively parallel approaches, and it’s not ideal for sequencing long genomes in a single run. Sanger sequencing analyzes DNA, not RNA, and it does determine the sequence of the DNA template. It isn’t limited to “cannot determine sequence”; the limitation is mainly how much sequence you can read in a single run and how quickly you can scale up the process.

Sanger sequencing uses chain-terminating dideoxynucleotides to create a set of DNA fragments that end at each possible base. When a ddNTP is incorporated, DNA polymerization stops because the molecule lacks the 3' OH needed to continue growth. By labeling these terminated fragments and separating them by size, you read off the sequence from the shortest to the longest fragment.

The main limitation is read length and throughput. Because you generate and read one fragment at a time per reaction lane, the readable stretch per run is relatively short (a few hundred bases in typical setups), and processing many fragments becomes time-consuming and labor-intensive. This makes Sanger sequencing low-throughput compared with newer, massively parallel approaches, and it’s not ideal for sequencing long genomes in a single run.

Sanger sequencing analyzes DNA, not RNA, and it does determine the sequence of the DNA template. It isn’t limited to “cannot determine sequence”; the limitation is mainly how much sequence you can read in a single run and how quickly you can scale up the process.