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Photostable Fluorescent dUTP Probes

AF488- and ATTO488-labeled dUTPs

Labeling of DNA probes for in situ hybridization is most often performed by enzymatic incorporation of labeled dUTP by PCR using Taq DNA polymerase[1-2]. More information…
We now offer linker-optimized AF488 (also known as Alexa Fluor® 488)-modified dUTP (dUTP-XX-AF488)
and ATTO488-XX-dUTP, providing photostable alternatives to traditionally used Fluorescein-12-dUTP (Fig. 1) (Tab. 1).

dTTP substitution
Figure 1: AF488-, ATTO-488 and Fluorescein-dUTPs label DNA very efficiently.
In the presence of intercalator dye (A) the entire formation of PCR product (labeled + unlabeled) is visualized. In the absence of intercalator (B) only dye-dNTP labeled PCR product is visible. For most PCR reactions the recommended dTTP substitution is 40 - 60 % however, some optimization may be required depending on individual assay conditions and final application of labeled probe. The optimal dTTP substitution for a specific PCR reaction can easily be determined by testing the whole range of 0 % - 100 % dTTP substitution by dye-modified dUTP.
L: 200 bp DNA Ladder; 0 % - 100 %: Increasing amount of dTTP substitution by dye-modified dUTP.

Table 1: Spectroscopic and substrate properties of ATTO488, AF488*- and Fluorescein-labeled dUTPs.
The choice of fluorophore depends on the final application and is a compromise between hydrophilicity and photostability that determine enzymatic incorporation efficiency and detection sensitivity, respectively. +: good, -: poor
Table 1 Spectroscopic and substrate properties of ATTO488, AF488 - and Fluorescein-labeled dUTPs.
* AF488 is also known as Alexa Fluor 488
** free acid

*** Please note: Optimal final concentration of the dye-labeled dUTP may vary depending on the application and assay conditions. For optimal product yields and optimal incorporation rates an individual optimization of the dye-labeled dUTP/ dTTP ratio is recommended.

Jena Bioscience is a primary manufacturer offering

  • both enzymatically incorporable Fluorescent, Hapten-, CLICK- or Amine-modified single nucleotides and corresponding kits
  • bulk amounts
  • custom formulations, packaging & labeling

Selected references:
[1] Morrison et al. (2003) Labeling Fluorescence In Situ Hybridization Probes for Genomic Targets. Methods in Molecular Biology 204:21.
[2] Wiegant et al. (1997) Probe Labeling and Fluorescence In Situ Hybridization. In: Current Protocols in Cytometry 8.3.1, John Wiley & Sons Inc.
Labeling of in situ hybridization DNA probes

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