Fluorescence In Situ Hybridization

Abstract

Abstract Fluorescence in situ hybridization (FISH) is a technique used to directly visualize specific DNA sequences on morphologically preserved cytological specimens such as metaphase chromosomes, interphase cell nuclei, and extended chromatin fibers or DNA molecules. The general usefulness of radioactive in situ hybridization was greatly hampered by the safety measures required when using radiolabeled probes and the rather long time (up to several weeks) needed for autoradiographic analysis. The development of powerful FISH protocols, in which unspecific hybridization of interspersed repetitive sequences contained within genomic DNA probes is suppressed, allowed for the first time the rapid visualization of single‐copy sequences and whole chromosomes, and thus marked a turning point in the experimental and diagnostic application of in situ hybridization. The major advantages of FISH over radioactive methods are increased spatial resolution, speed, probe stability, and the ability to detect multiple chromosomal targets in different colors. Indirect FISH uses hapten‐labeled DNA probes in combination with immunocytochemistry (secondary detection reagents). Direct methods use fluorochromes directly coupled to nucleotides in the probe DNA. Probe labeling is most easily accomplished by nick translation or other enzymatic labeling procedures. For hybridization of the modified probe DNA to chromosomes and cell nuclei, both probe and target DNA must be denatured. In the hybridization reaction, complementary single‐stranded sequences in the probe and chromosomal target are allowed to reanneal. After posthybridization washing and immunocytochemical detection of in situ bound hapten molecules, a specific fluorescent signal is produced at the hybridization site, which can be viewed by eye through the epifluorescence microscope. Digital imaging and computerized storage of images have largely replaced photography and greatly facilitated data analysis and image handling. However, most importantly the success of FISH experiments depends on the quality of the chromosome preparations and the size of the chromosomal DNA target. When large‐insert (>30 kb (kilobase)) clones or complex DNA libraries are used as FISH probes, between 80% and 100% of the target chromosomes carry visible hybridization signals. This percentage drops to only a few percent when 1 kb or less of a single‐copy sequence is hybridized. In the past few years several technical advances have expanded FISH applications. Comparative genomic hybridization (CGH) of differentially labeled tester (tumor or patient) and normal reference DNA allows the analysis of all genetic imbalances (chromosomal gains and losses) in a tester genome within a single experiment and without the need to prepare chromosomes from the tester. Combinatorial probe labeling and multicolor FISH are used for the simultaneous detection of 24 (or more) chromosomal targets in different colors, and therefore can provide a comprehensive picture of extensively rearranged tumor karyotypes. The DNA resolution of FISH has been dramatically increased (from several megabases (Mbs) to several kilobases) by hybridization to extended chromatin fibers and linearized DNA molecules. Fiber FISH and molecular combing allow one to order probes relative to each other, to orient probes within a contig, and to determine the degree of overlap or gap size between different probes. Overall, FISH has become an increasingly popular method with a broad spectrum of applications in cytogenetics, genomics, tumor biology, and many other research areas. It has a bright and colorful future.