Quenched Nucleotide Fluorescent Reporters
Several methods have been developed for enzymatic fluorescent labelling of nucleic acids. A dNTP analog can be used to incorporate a fluorophore by PCR, nick translation or random priming, either directly into DNA1 or indirectly via a hapten such as biotin.2 Though high incorporation efficiencies have been reported,3 all of these approaches require the separation of unincorporated label prior to downstream applications. A reagent called an Internally Quenched Nucleotide or IQN has been developed by Lawler Scientific, LLC. This reagent consists of a nucleoside triphosphate with a fluorescent reporter attached to the nucleobase and a quencher moiety attached to the gamma-phosphate. The nucleotide remains non-fluorescent until the quencher is enzymatically separated from the parent nucleotide. Since IQNs are non-fluorescent until incorporated into a nucleic acid, they do not give rise to the background fluorescence signals commonly observed when DNA labelled by standard means is inadequately purified.
The first generation IQN consists of a fluorescein-dUTP with a dabsyl quencher linked to the gamma phosphate. Fluorescein and dabsyl were selected because of their superior optical properties and because the photophysics governing their interaction is well described in the literature.4 In addition, this IQN is soluble and stable in aqueous solution.
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and A.S. Waggoner, Nucleic Acids Res., 1994, 22, 3226-32.
(2) X. Li, W.M. James, F. Traganos, and Z. Darzynkiewicz, Biotech Histochem,
1995, 70, 234-42.
(3) T. Tasara, et al., Nucleic Acids Res., 2003, 31, 2636-46.
(4) S.A.E. Marras, F.R. Kramer, and S. Tyagi, Nucleic Acids Res., 2002,
(5) D.A. Berry, et al., Tetrahedron Lett, 2004, 45, 2457-2461.