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#ddNTPs

2 public questions tagged with this topic.

Sequencing

What is missing in ddNTPs that causes chain termination?

Dideoxynucleotides are analogs of deoxynucleotides that lack hydroxyl groups at both the 2' and 3' positions of the ribose sugar. Deoxynucleotides lack only the 2' OH, retaining 3' OH essential for chain elongation. DNA polymerase catalyzes nucleophilic attack of the primer 3' OH on the alpha-phosphate of incoming nucleotide. When a ddNTP is incorporated, no 3' OH remains to attack the next nucleotide, so phosphodiester bond formation cannot continue. Consequently, chain growth aborts. Missing base or 5' OH does not explain termination; absence of 3' OH is critical.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Sequencing

Which nucleotide analogs are used in Sanger sequencing?

Sanger sequencing employs dideoxynucleoside triphosphates, ddNTPs, as essential terminators alongside normal dNTPs. The reaction contains all four dNTPs for strand extension plus limiting concentrations of labeled ddATP, ddTTP, ddGTP and ddCTP in four separate or single combined reactions. DNA polymerase synthesizes complementary strand until a ddNTP is randomly incorporated. Since ddNTPs lack a 3' hydroxyl group, further phosphodiester bond formation is impossible and synthesis halts permanently. Ribonucleotides rNTPs are not used, and dATP alone is insufficient for comprehensive sequence determination.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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