Fluorescence labels, distinguishing

The labeling can also be done by fluorescence lifetime differences, e.g., introduced by quenching pathways connected with different surroundings. This can be used for lifetime imaging methods (Chapter 1, this volume) or for distinguishing complexes of one and the same fluorescence label with, e.g., different DNA-bases. In this case, the fluorescence label is not only a label but incorporates a function which senses the environment and can therefore be regarded as a sensing fluorescence probe. 

Microsatellites are hypervariable co-dominant loci composed of arrays of 2-9 bp repeating motifs. Differences in the number of repeat motifs in an array define microsatellite polymorphisms. Method development requires the identification of microsatellite loci, and for each locus, the design of PCR primers to anneal to conserved regions flanking the microsatellite. Analysis involves PCR amplification with fluorescently labeled primers followed by electrophoresis to distinguish microsatellite alleles of different array size. 

Cell proliferation can be measured by different techniques in various in vitro models and is used as one of the most specific endpoints for DNT evaluation. A commonly used assay is the bromo-deoxyuridine (BrdU) incorporation where analogues nucleotides are integrated into newly synthesized DNA permitting indirect detection of rapidly proliferating cells by fluorescent labelled anti-BrdU antibodies [49], Cytotoxicity assays, such as MTT and ala-marBlue, are also sometimes used to measure proliferation [49]. However, these assays do not distinguish between cell death and inhibited cell proliferation which is of particular importance in DNT studies. 

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.]... 

A similar but smaller microarray of gel-immobilized, fluorescence-labeled nucleic acids is being developed by Argonne National Laboratory (Yershov et al., 1996). One application seeks to develop a "bacillus microchip" that will detect B. anthracis, indicate whether it is alive or dead (DNA matches, but no RNA matches), and distinguish it from other related bacteria, such as B. thuringiensis, B. subtilis, and B. cereus (Mirzabekov, 1998). 

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