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#nucleotide incorporation

3 public questions tagged with this topic.

Replication Basics

Incorrect nucleotide incorporation is reduced mainly by

Even highly selective polymerases occasionally incorporate non-complementary nucleotides at rates near 10^-5 during rapid synthesis. Immediate correction relies heavily on intrinsic 3' to 5' exonuclease proofreading activity located in separate domain. When misincorporation distorts primer-template duplex geometry, polymerase stalls and transfers frayed 3' end from polymerase active site to exonuclease site that hydrolyzes phosphodiester bond and excises incorrect base. Strand then repositions into polymerase site for continued accurate synthesis. This kinetic proofreading step improves fidelity about 100-fold reducing errors to 10^-7 before mismatch repair.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 11: DNA replication fidelity and proofreading exonuclease

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Sequencing

Ion Torrent sequencing detects:

Ion Torrent semiconductor sequencing, introduced by Life Technologies, determines sequence without optics. Each microwell contains template-coated beads and a polymerase. When a deoxynucleotide complementary to template is incorporated, a covalent phosphodiester bond forms and a proton, hydrogen ion, is released as byproduct. This release transiently acidifies microwell solution, typically by 0.02 pH units, detected by an ion-sensitive field-effect transistor beneath well. Signal amplitude reflects homopolymer length. Detection principle is therefore pH change, not light, electrical charge of DNA itself or fluorescence employed in other platforms.

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 sequencing method produces light after nucleotide incorporation?

Pyrosequencing is a non-electrophoretic sequencing-by-synthesis platform that couples nucleotide incorporation to visible light production. Each cycle dispenses a single dNTP; if it is complementary to next template base, DNA polymerase incorporates it and releases inorganic pyrophosphate, PPi, stoichiometrically. ATP sulfurylase converts PPi to ATP, which drives firefly luciferase to oxidize luciferin emitting photons. Light intensity indicates incorporation and number of same bases in homopolymer runs. Sanger uses termination fluorescence, Ion Torrent measures pH change, and Maxam-Gilbert relies on chemical cleavage without light.

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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