Fluorescently labeled probes

Total RNA is isolated from the lymphocytes according to standard procedures and used as a template for radioactive labeled cDNA synthesis. The purified cDNA is used as probe for cDNA expression arrays. The advantages of this method as compared to other array systems are as follows (1) Radioactive-labeled probes are more sensitive than fluorescent-labeled probes and therefore need less sample RNA. (2) The primers used in the cDNA synthesis match the genes represented on the array. (3) The primer sequences are longer compared to other array systems, which increases the hybridization fidelity of RNA to the matching correct set of genes and therefore reduces mismatch reactions. 

Thiazole orange derivatives have also been covalently attached to oligonucleotides to generate a fluorescently labelled probe oligonucleotide capable... 

Luo Y, Knuckley B, Bhatia M et al (2006) Activity-based protein profiling reagents for protein arginine deiminase 4 (PAD4) synthesis and in vitro evaluation of a fluorescently labeled probe. J Am Chem Soc 128 14468-14469... 

PBPs). A group at Lilly has introduced the radiolabeled probe 56 and fluorescence-labeled probe 57 as tool compounds to profile the PBPs in different bacterial strains using SDS-PAGE (Fig. 26) [100, 101]. At this time, no MS analysis of the protein bands has been performed. 

Moreover, combination of a fluorine-containing linker and a fluoro-taxoid may provide fluorine probes for monitoring the internalization and drug release of these conjugates in the tumor cells and tissues by 19F NMR as an alternative method to the use of fluorescence-labeled probes with confocal fluorescence microscopy [69],... 

A fluorescence in situ hybridization (FISH) technique, UroVysion, uses fluorescently labeled probes to detect aneu-ploidy of chromosomes 3,7, and 17, and deletion of the 9p21 locus that contains the tumor suppressor pi6, which is the most common alteration seen in urothelial carcinoma. Table 23-13 compares cytology with that of UroVysion for the detection of bladder cancer. 

HPLC is commonly used for separating and purifying oligonucleotides. Separation is usually based on ion-pair, reversed-phase chromatography and is particularly useful for purifying fluorescently labeled probes guided by absorbance and fluorescent elution profiles. 

The four simplest methods do not even use fluorescently labeled probes. Amplicon melting uses only two primers and a double-strand DNA dye (see Figure 37-30, row three). 

Another hybridization probe design includes a single fluorescently labeled probe [4]. In this case, fluorescence is transferred by a G on the opposite strand, but adjacent to the labeled end of the probe. Fluorescence is... 

During the FISH procedure, DNA within cells placed on a slide and the fluorescently labeled probe of interest are denatured by incubation at high temperature. Then the probe is allowed to hybridize to the target DNA. The next step is a series of post-hybridization washes to remove the probe excess. Finally, after counterstaining the nuclei, the probe signal can be visualized under a fluorescent microscope. 

In situ hybridization experiments with several different fluorescent-labeled probes to DNA in human Interphase cells support the loop model shown in Figure 10-24. In these experiments, some probe sequences separated by millions of base pairs in linear DNA appeared reproducibly very close to one another in interphase nuclei from different cells (Figure 10-25). These closely spaced probe sites are postulated to lie close to specific sequences in the DNA, called scaffold-associated regions (SARs) or matrix-attachment regions (MARs), that are bound to the chromosome scaffold. SARs have been mapped by digesting histone-depleted chromosomes with restriction enzymes and then recovering the fragments that are bound to scaffold proteins. 

A EXPERIMENTAL FIGURE 10-25 Fluorescent-labeled probes hybridized to interphase chromosomes demonstrate chromatin loops and permit their measurement. In situ hybridization of interphase cells was carried out with several different probes specific for sequences separated by known distances in linear, cloned DNA. Lettered circles represent probes. Measurement of the distances between different hybridized probes, which could be distinguished by their color, showed that some sequences (e.g.. A, B, and C), separated from one another by millions of base pairs, appear located near one another within nuclei. For some sets of sequences, the measured distances in nuclei between one probe (e.g., C) and sequences successively farther away initially appear to increase (e.g., D, E, and F) and then appear to decrease (e.g., G and H). The measured distances between probes are consistent with loops ranging in size from 1 million to 4 million base pairs. [Adapted from H. Yokota et al., 1995, J. Cell Biol. 130 1239.]... 

An improved delivery scheme for intracellular tracking and anticancer therapy uses a novel double functionalization of a carbon nanotube delivery system containing antisense oligodeoxynucleotides as a therapeutic gene and CdTe QDs as fluorescent labeling probes via electrostatically layer-by-layer assembling [39]. 

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