Question

Which sequencing technique relies on the synthesis of complementary strands in the presence of chain-terminating dideoxynucleotides?

• A. Sanger sequencing
• B. Next-generation sequencing
• C. Polymerase Chain Reaction (PCR)
• D. Pyrosequencing

Hint:

• Sanger Sequencing (Dideoxy / Chain-Termination Method)
  • Uses DNA polymerase, template, primer, dNTPs, and chain-terminating ddNTPs.
  • ddNTPs lack a 3'-OH group, so their incorporation stops DNA synthesis.
  • Generates a set of DNA fragments of different lengths, each ending with a specific ddNTP.
• This method was the dominant DNA sequencing technology for decades.

Answer 1

The Correct Option is A

The sequencing technique described is Sanger sequencing, also known as the chain-termination method or dideoxy sequencing method. Key principles of Sanger sequencing:
1. It involves in vitro DNA synthesis of a complementary strand using a DNA template, a primer, DNA polymerase, normal deoxynucleotide triphosphates (dNTPs: dATP, dGTP, dCTP, dTTP), and a small amount of chain-terminating dideoxynucleotide triphosphates (ddNTPs: ddATP, ddGTP, ddCTP, ddTTP).
2. Dideoxynucleotides (ddNTPs) lack the 3'-hydroxyl (3'-OH) group that is necessary for the formation of a phosphodiester bond with the next incoming nucleotide.
3. When a ddNTP is incorporated by DNA polymerase into a growing DNA strand, synthesis of that strand is terminated at that point.
4. By running four separate reactions, each containing a different ddNTP (or using fluorescently labeled ddNTPs in a single reaction for automated sequencing), a set of DNA fragments of varying lengths is generated, each terminated at a specific base.
5. These fragments are then separated by size (e.g., by gel electrophoresis or capillary electrophoresis), and the sequence is read from the order of the terminated fragments. Let's analyze the options: (a) Sanger sequencing: This directly matches the description. (b) Next-generation sequencing (NGS): A broad term for several high-throughput sequencing technologies (e.g., Illumina sequencing, Ion Torrent sequencing, PacBio SMRT sequencing). While they involve DNA synthesis, their core mechanisms are different from dideoxy chain termination (e.g., sequencing by synthesis with reversible terminators, sequencing by ligation, single-molecule real-time sequencing). (c) Polymerase Chain Reaction (PCR): Used to amplify DNA, not primarily for sequencing (though PCR products are often sequenced). (d) Pyrosequencing: A sequencing-by-synthesis method that detects the release of pyrophosphate (PPi) upon nucleotide incorporation. It does not use chain-terminating dideoxynucleotides.
Therefore, Sanger sequencing relies on chain-terminating dideoxynucleotides. \[ \boxed{\text{Sanger sequencing}} \]