Digoxigenin-11-dUTP is enzymatically incorporated into DNA/cDNA as substitute for its natural counterpart dTTP. The resulting Digoxigenin-labeled DNA/cDNA probes are subsequently detected using Digoxigenin-antibodies conjugated with horseradish peroxidase (HRP), alkaline phosphatase (AP) or a fluorescent dye. Optimal substrate properties and thus labeling efficiency is ensured by an 11-atom linker attached to the C5 position of uridine.
Digoxigenin-11-dUTP can be incorporated into DNA/cDNA by
- PCR with Taq polymerase
- Nick Translation with DNAse I/ DNA Polymerase I
- Primer Extension with Klenow exo-
- 3’-End Labeling with Terminal deoxynucleotidyl Transferase (TdT)
- Reverse Transcription with MMLV Reverse Transcriptase
Digoxigenin-11-dUTP can be incorporation into RNA by
- 3’-End Labeling with Terminal deoxynucleotidyl Transferase (TdT)
Recommended Digoxigenin-11-dUTP/dTTP ratio for PCR and Nick Translation: 35% Digoxigenin-11-dUTP/ 65% dTTP
Please note: The optimal final concentration of Digoxigenin-11-dUTP may depending on the application and assay conditions. For optimal product yields and high incorporation rates an individual optimization of the Digoxigenin-11-dUTP/dTTP ratio is recommended
References:
[1] Anderson et al. (2005) Incorporation of reporter-labeled nucleotides by DNA polymerases. Biotechniques 38:257.
[2] Jackson et. al (1991) Detection of Shiga Toxin-Producing Shigella dysenteriae Type 1 and Escherichia coli by Using Polymerase Chain Reaction with Incorporation of Digoxigenin-11-dUTP. J Clin Microbiol. 29 (9):1910.
[3] Dauwerse et al. (1999) Two-colour FISH detection of the inv (16) in interphase nuclei of patients with acute myeloid leukemia. Br J Haematol 106:111.
[4] Wiegant et al. (2008) Probe Labeling and Fluorescence In Situ Hybridization. Current Protocols in Cytometry: Unit 8.3.
[5] Schmitz et al. (2008) Nonradioactive labeling of oligonucleotides in vitro with the hapten digoxigenin by tailing with terminal transferase. Anal Biochem 199:222.
[6] Grimmond et al. (2001) Expression Profiling with cDNA Microarray's: A User's Perspective and Guide. In: DNA Microarrays: Gene Expression Applications (Jordan). Springer Verlag Berlin Heidelberg.
[7] Rosemeyer et al. (1995) Nonradioactive 3'-end-labeling of RNA molecules of different lengths by terminal deoxynucleotidyltransferase Anal Biochem 224:446.