A chemical method for fast and sensitive detection of DNA synthesis <i>in vivo</i>

TLDR

The method detects DNA synthesis by incorporating 5‑ethynyl‑2′‑deoxyuridine (EdU) into replicating DNA and subsequently labeling it with a fluorescent azide via a Cu(I)-catalyzed click reaction. The EdU‑click assay is highly sensitive, rapid, penetrates large tissues, preserves structure without fixation or denaturation, and works in cultured cells as well as in the intestine and brain of whole animals.

Abstract

We have developed a method to detect DNA synthesis in proliferating cells, based on the incorporation of 5-ethynyl-2′-deoxyuridine (EdU) and its subsequent detection by a fluorescent azide through a Cu(I)-catalyzed [3 + 2] cycloaddition reaction (“click” chemistry). Detection of the EdU label is highly sensitive and can be accomplished in minutes. The small size of the fluorescent azides used for detection results in a high degree of specimen penetration, allowing the staining of whole-mount preparations of large tissue and organ explants. In contrast to BrdU, the method does not require sample fixation or DNA denaturation and permits good structural preservation. We demonstrate the use of the method in cultured cells and in the intestine and brain of whole animals.