Probes fluorochrome labeling

T4 RNA ligase can introduce fluorescent 3 -O-(5 -phos-phoryldeoxycytidyl) S-bimane phosphorothioate into RNA or DNA (Cosstick et al., 1984). Other methods have been developed for fluorochrome labeling of DNA for automated sequencing, either by labeled primers or dye-terminators (fluorescent ddNTP Applied Biosystems) with DNA polymerase or with terminal dNTP transferase (Trainor and Jensen, 1988). We successfully used fluorescein-dUTP (Boehringer Mannheim, soon available), both for automated sequencing and probe preparation. All common DNA polymerases incorporate this analogue efficiently. 

Direct method in which the probe-target hybrid can be visualized after the hybridization. When fluorochrome-labeled probes are used, the visualization can be achieved by fluorescence microscopy. Various fluorochromes with different emission colors are now available such as AMA, FITC, fluorescein, rhodamine CY3 and Texas red. The use of different probes labeled by different fluorochromes... 

Direct Fluorochrome-Labeled DNA Probes for Direct Fluorescent In Situ Hybridization to Chromosomes... 

Chromosomal in situ hybridization enables determination of the presence and location of DNA sequences complementary to a labeled probe along chromosomes and within interphase nuclei. The use of directly fluorochrome-labeled probes means that sites of probe hybridization can be directly visualized. Lengthy detection procedures, as in the indirect immunochemistry methods to detect biotin or digoxigenin, are not needed. However, because there is no signal amplification, sensitivity is limited and the system is used mostly to detect middle- to high-copy number (Fig. 1) or pooled probes, although antibody amplification of some fluorochrome labels is possible (1), and then the method must be evaluated in comparison with digoxigenin (Chapter 27) or biotin (Chapter 25). 

Probe Label the probe DNA with direct fluorochrome-labeled nucleotides (available from Amersham [Amersham, UK] or Boehringer Mannheim [Mannheim, Germany]) as described in Chapter 15. Incor-... 

Fluorochromes are visualized by excitation with light of one (excitation) wavelength and imaging emitted fluorescence at another (emission) wavelength using appropriate filters. The properties and methods of visualization of the fluorochromes fluorescein and rho-damine, which are used to detect digoxigenin, are the same as for direct fluorochrome-labeled probes and are outlined in Chapter 26. 

Young, D.M., Greulich, K.M., and Weier, H.G., Species-specific in situ hybridization with fluorochrome-labeled DNA probes to study vascularization of human skin grafts on athymic mice, /. Burn Care Rehabil, 17,305,1996. 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

Immunocytochemistry The presence of the labeled probe bound to the in situ nucleic acid target is detected by means that relate specifically to the nature of the label. The most frequently used labels are enzymes (alkaline phosphatase, hydrogen peroxidase) and fluorochromes (fluorescein, rhodamine, hydroxycoumarin). 

Fig. 7.4, Fluorochromes used as labels in probes (I—II). Fluorescent dye (III) is used as a substrate, whereas Eu (IV) is used in time-resolved fluorescence.

Alexa fluorochromes are available only as a protein labeling kit from Invitrogen, Molecular Probes, Inc. (Carlsbad, CA, USA www.probes.com). The reactive dye has a succinimidyl ester moiety that reacts with primary amines of proteins. The conjugation steps are similar to those for fluorescein isothiocyanate. 

Figure 16.2. The process of microarray hybridization using printed DNA probes. A robotic printer deposits DNA in a regular array on a series of glass slides. After they are processed, the slides are hybridized to a mixture of two cDNA pools derived from test and reference samples that have been labeled with spectrally distinct fluorochromes. After stringency washes, the microarray is scanned in a laser-scanning device, and the image is processed to generate numerical data. (See color plate.)...

Direct visualization is performed by fluorescence microscopy when fluoro-chrome-labeled probes or florescence-labeled antibodies are used (fluorescence in situ hybridization, FISH). The color of fluorescence depends on the type of used fluorochrome, blue (AMCA), green (fluorescein) and red (rhoda-mine, Texas red, CY3 and TRITC). 

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