The powerful functionality of 5-Ethynyl dUTP stems from the underlying "click chemistry" principle. This is a highly efficient, specific, and biocompatible synthetic reaction, hailed as the "Lego of the molecular world".

Its workflow is divided into two steps, ingenious and reliable:

Step 1: Incorporation into DNA

In cell culture systems or in vitro enzymatic reactions (such as PCR, sequencing), 5-Ethynyl dUTP is used as a raw material by DNA polymerase, replacing part of the natural dTTP, and directly integrated into the interior of nascent DNA molecules.

Step 2: Click Conjugation

The DNA incorporating the ethynyl group can undergo a copper-catalyzed cycloaddition reaction with reporter molecules bearing an azide group (-N3), such as fluorescent dyes (e.g., Alexa Fluor series) or biotin.

This reaction is rapid, efficient, and has extremely low background, forming a stable covalent linkage under mild conditions, thereby enabling fluorescence imaging, flow cytometry detection, or affinity enrichment of the labeled DNA.