How does Sanger sequencing determine DNA sequence, and what is the role of ddNTPs?

How does Sanger sequencing determine DNA sequence, and what is the role of ddNTPs?

• A. It resolves chain-terminated fragments produced when ddNTPs are incorporated, enabling readout of sequence by labeled fragments.
• B. It sequences DNA by parallel synthesis of many fragments and uses ddNTPs to label each fragment's end.
• C. It is based on sequencing-by-ligation to determine bases with ddNTP terminators.
• D. It uses ddNTPs to elongate the chain and read sequence.

Answer: (A)

At the heart of Sanger sequencing is using dideoxynucleotides to create a set of DNA fragments that all stop growing at the point where a specific base is incorporated. DNA polymerase extends a primer, adding normal nucleotides, but occasionally a ddNTP is incorporated instead. Because ddNTPs lack the 3' hydroxyl group needed to form the next phosphodiester bond, their incorporation terminates the chain. If ddNTPs are present in small amounts compared with normal nucleotides, you get a mixture of fragments that end at each possible base, producing a ladder of terminated fragments that differ by one nucleotide in length. Each fragment ends with a labeled ddNTP, so when you separate the fragments by size (usually by capillary electrophoresis) you can read the terminating base from the label and reconstruct the sequence by ordering the fragments from shortest to longest. This termination mechanism is what makes Sanger sequencing readable base by base. Other methods rely on different principles (for example, sequencing by synthesis with parallel approaches or sequencing by ligation) and do not use ddNTPs to terminate chains in the same way.

At the heart of Sanger sequencing is using dideoxynucleotides to create a set of DNA fragments that all stop growing at the point where a specific base is incorporated. DNA polymerase extends a primer, adding normal nucleotides, but occasionally a ddNTP is incorporated instead. Because ddNTPs lack the 3' hydroxyl group needed to form the next phosphodiester bond, their incorporation terminates the chain. If ddNTPs are present in small amounts compared with normal nucleotides, you get a mixture of fragments that end at each possible base, producing a ladder of terminated fragments that differ by one nucleotide in length. Each fragment ends with a labeled ddNTP, so when you separate the fragments by size (usually by capillary electrophoresis) you can read the terminating base from the label and reconstruct the sequence by ordering the fragments from shortest to longest. This termination mechanism is what makes Sanger sequencing readable base by base. Other methods rely on different principles (for example, sequencing by synthesis with parallel approaches or sequencing by ligation) and do not use ddNTPs to terminate chains in the same way.