Direct Versus Indirect Methods

In direct in situ hybridization, the fluorescent reporter molecule is bound to the nucleic acid probe so that hybrids that have formed can be visualized microscopically immediately after in situ hybridization (Wiegant et al., 1991). In indirect procedures, the probe contains an element that renders it detectable by additional labeling steps (e.g., biotin-streptavidin binding or immunocytochemistry): hence the term "indirect." A number of such hapten modifications have been described. Direct methods are also amenable to immunocytochemical amplification if antibodies against the reporter molecules are available (Raap et al., 1990; Wiegant et al., 1991).

Haptens currently in use include biotin, di-goxigenin, and fluorescein. Fluorescein tetramethyl rhodamine, aminomethyl coumarin acetic acid (AMCA), and a series of cyanin dyes are in widespread use as fluorochromes, providing good spectral coverage across visual and infrared wavelengths. Although chemical methods for DNA labeling exist, generally haptens and fluorochromes are incorporated enzymati-cally into newly synthesized DNA using hapten-or fluorochrome-modified dUTP. The allyla-mine derivative of dUTP can be fluorochrom-ized or haptenized, for example using V-hy-droxysuccinimide esters of haptens and fluorochromes. Its use for enzymatic synthesis of nonradioactive probes (Langer et al., 1981) was a major achievement because it fit closely with existing molecular biology formats for the radioactive labeling of nucleic acids employing DNA polymerases (e.g., by nick translation or random-primed labeling). This achievement led to the widespread application of nonra-dioactive probes in in situ hybridization.

Fluorescence in situ hybridization (FISH) methods have achieved high standards of sensitivity, resolution, and multiplicity. In the following sections, these parameters and the conditions under which they are obtained are presented, and molecular cytogenetic applications of FISH are briefly discussed.

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