A common approach for DNA synthesis monitoring relies on incorporation of ethynyl-labeled deoxyuridine (5-EdU) instead of its natural counterpart thymidine[1-5]. Labeling of the resulting ethynyl-functionalized DNA is achieved via Copper (Cu(I))-catalyzed Azide-Alkyne Click chemistry reactions (CuAAC) that allows to introduce a fluorescent group for subsequent microscopic imaging.
The Singh Lab developed a step-by-step protocol for DNA synthesis monitoring in Zebrafish which they used as model organism to study liver biology (Fig. 1). This protocol consists of a four-step procedure:
Figure 1: De novo DNA synthesis in Zebrafish is visualized by 5-EdU-based Copper-catalyzed Click Chemistry.
Zebrafish larvae were treated with 10mM 5-EdU at 4-days post-fertilization (dpf). At 5 dpf (24 hour chase), the larvae were fixed for staining using the CuAAC Cell Reaction Buffer Kit (BTTAA based) and AZDye647-Picolyl-Azide according to the step-by-step protocol.
Transgenic lines used: Tg(fabp10a:H2B-mGL) & Tg(tp1:H2B-mCherry)
Hepatocytes: green
Notch-responsive cholangiocytes: red
5-EdU: white
| Product | Cat. No. | Amount | Price |
|---|---|---|---|
| 5-Ethynyl-2'-deoxyuridine (5-EdU) | CLK-N001-25 | 25 mg | 69,40 € |
| CLK-N001-100 | 100 mg | 201,60 € | |
| CLK-N001-500 | 500 mg | 639,70 € | |
| CLK-N001-5000 | 5 g | 3.358,40 € | |
| CuAAC Cell Reaction Buffer Kit (BTTAA based) | CLK-073 | 1 kit | 307,40 € |
| AZDye647-Picolyl-Azide | CLK-1300A-AZ-1 | 1 mg | 219,00 € |
| CLK-1300A-AZ-5 | 5 x 1 mg | 564,00 € |
Please contact Barbara with all questions or inquiries you may have!
Contact Barbara
[1] Salic et al. (2008) A chemical method for fast and sensitive detection of DNA synthesis in vivo. Proc. Natl. Acad. Sci. USA 105:2415.
[2] Li et al. (2010) Fluorogenic "click" reaction for labeling and detection of DNA in proliferating cells. Biotechniques 49 (1):525.
[3] Diermeier-Daucher et al. (2009) Cell Type Specific Applicability of 5-Ethynyl-2'-deoxyuridine (EdU) for Dynamic Proliferation Assesment in Flow Cytometry. Cytometry A 75A:535.
[4] Darzynkiewicz et al. (2011) Cytometry of DNA Replication and RNA synthesis: Historical Perspective and Recent Advances Based on "Click Chemistry". Cytometry A 79A:328.
[5] Diermeier-Daucher et al. (2010) Dynamic Proliferation Assessment in Flow Cytometry. Curr. Protoc. Cell Biol. 48:8.6.1.
