Fluorescent oligo

Quantitative PCR without using an internal standard has become possible with the recent devdopment of real-time thermocyclers. The advantage of this approach is that the amplified DNA is detected in the exponential phase of the amplification rather than the plateau phase, by sensitive on-line methods using fluorescent oligo-nudeotide probes or fluorescent dyes (Meuer et al. 2001). 

Fig. 10 Confocal laser scanning microscope images of islets with urokinase (UK) immobilized on the membrane. The green fluorescence indicates positive immunostaining for UK. (a) Islets were modified with oligo(dT)2o-PEG-lipid (C16) or (b) oligo(dT)2o-PEG-lipid (C18) then, oligo (dA)2o-UK was added to the media, (c) Unmodified islets with (left) and without (right) oligo (dT)20-PEG-lipids added to the solution. Insets. Bright field images. Scale bars 100 pm...

Reduction of the cystamine-labeled oligo using a disulfide reducing agent releases 2-mer-captoethylamine and creates a thiol group for conjugation (Figure 27.6). DNA probes labeled in this manner have been successfully coupled with SPDP-activated alkaline phosphatase (Chapter 26, Sections 1.2 and 2.5), maleimide-activated horseradish peroxidase (HRP) (Chapter 26, Section 1.1), NHS-LC-biotin (Chapter 11, Section 1 and Chapter 27, Section 2.3), and the fluorescent tag AMCA-HPDP (Chapter 9, Section 3 and Chapter 27, Section 2.5). 

Purify the SH-labeled oligo by gel filtration on a desalting resin using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. The probe now may be used to conjugate with an activated enzyme, biotin, fluorescent tag, or other molecules containing a sulfhydryl-reactive group. 

Fluorescent probes containing sulfhydryl-reactive groups can be coupled to DNA molecules containing thiol modification sites. The chemical derivatization methods outlined in Section 2.2 (this chapter) may be used to thiolate the oligo for subsequent modification with a fluorophore. Appropriate fluorescent compounds and their reaction conditions may be found in Chapter 9. The protocol discussed in the previous section can be used as a general guide for labeling DNA molecules. 

Another series of GSL mimics with oligo-ethylene glycol as spacer have also been obtained successfully using trichloroacetimidate method [482]. In addition, fluorescence-labeled sLex glycosphingolipids have also been chemically synthesized as targets for investigating microdomain formation in membranes [483]. 

In a totally independent approach, highly fluorescent but rigid molecules can be used and their reorientational movement probed in solution, mostly by time-resolved fluorescence anisotropy, (85) For rodlike molecules likep-oligo-phenyls, this movement corresponds to the reorientation of the long molecular axis the length of which can be easily varied. 86 The use of fluorescence probes in liquid and solid media and on surfaces has been treated recently in several reviews and books. 87 ... 

Probes can be differently labeled with hapten labels, for example carboxyfluorescein (6-FAM), digoxigenin (DIG) and biotin can be bound to LNA oligos. The choice of probe label depends on experimental design and the techniques available in the laboratory. The hapten label provides a template for crucial signal amplification since the FITC label on the oligo itself is not sufficient to allow detection in standard epifluorescence. In this study, the fluorescence signal was obtained with the TSA-FITC substrate, which allowed detection of miR-21 and miR-205. 

Some other 3-thienyl ether derivatives have been prepared. Poly[3-oligo(oxyethylene)-4-methyltiophene] showed an absorption peak at 550 nm (95JCS(CC)2293). On the other hand, poly[3-(N-succinimido(tetra-ethoxy)oxy)-4-methylthiophene] was prepared and it is able to react with amines to give the corresponding carbamate with a 15-crown-5 attached substructure. This polymer showed absorption at 429 nm and fluorescence at 543 nm (04T11169). 

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