Oligonucleotide fluorescein

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

Oligonucleotides bearing nido-carborane have been synthesized as phospho-diesters using an automated DNA synthesizer (see Scheme 2.2-14) [39]. These oligophosphates are homogeneous, very hydrophilic and are readily taken up into cells. Fluorescein-labeled nido-carboranyl oligomeric phosphate diesters accumulate in the cell nucleus [40]. 

Fig. 2. Gene mapping by PRINS Three sets of oligonucleotides were used to map the COL9A2 gene on chromosome lp32. The signal was initially detected with avidin fluorescein and enhanced with antifluorescein peroxidase followed by DAB/gold sil-ver as described in the text.

Fig. 6. Electropherogram of fluorescein labeled phosphorothioate oligonucleotides (PS pd (T)io-25) recorded in 10% T,0% C polyacrylamide matrix at 2000 V/cm,L = 38 mm (reprinted with permission from [23]. Copyright 1994 American Chemical Society).

FIGURE 4.3 Repetitive sample injection and separation. Cycle conditions injection time, 5 s dead time, 1 s separation time, 45 s dead time, 1 s. The insets show two separation events on an expanded time scale. Sample fluorescein-labeled phosphorothioate oligonucleotide mixture, poly(dT)10 25. Separation conditions buffer, 100 mM Tris, 100 mM boric acid, 2 mM EDTA, 7 M urea, pH 8.5 Electric field strength, 2300 V/cm separation length, 3.8 cm [547]. Reprinted with permission from the American Chemical Society. 

Free-solution (gel-free) CE separation has been used in the detection of a target gene sequence amplified using the CPT reaction (see Chapter 9, section 9.2.1.7 for details). The intact chimeric oligonucleotide probe (fluorescein-labeled at the 5 end and biotin-labeled at the 3 end) was separated from a cleaved probe (only fluorescein-labeled at the 5 end). Owing to the presence of biotin in the intact probe, it migrated later than the cleaved probe, even in the absence of a gel sieving matrix [606]. 

Zhang, J., Malicka, J., Gryczynski, I., and Lakowicz, J. R. (2005). Surface-enhanced fluorescence of fluorescein-labeled oligonucleotides capped on silver nanoparticles, yowrno/ of Physical Chemistry B 109 7643-7648. 

Figure 20.6 Schematic diagram of the DNA detection using fluorescence quenching by gold nanoparticles. Oligonucleotide molecules are conjugated to gold surface vial Au-S bonds and the fluorescein molecules at the other end of the oligo sequence is adsorbed onto gold nanoparticles to form a constrained conformation.

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