Fluorescence In Situ Hybridization

An opportunity to look inside of the individual cell for the direct visualization in situ of "what happened?" is the most wonderful feature offered by fluorescence in situ hybridization (FISH). DNA in situ hybridization is a technique that allows the visualization of defined sequences of nucleic acids within the individual cells. The method is based on the site specific annealing (hybridization) of single-stranded labeled DNA fragments (probes) to denatured, homologous sequences (targets) on cytological preparations, like metaphase chromosomes, interphase nuclei, or naked chromatin fibers. Visualization of hybridization sites becomes possible after detection steps by using a wide spectrum of the fluorescent dyes available.

Much has been achieved during the last decade in the human genome analysis due to the development of nonradioactive methods of DNA in situ hybridization. General usefulness of FISH for physical mapping (1-2) was greatly enhanced by improved DNA resolution. Interphase cytogenetics has become an useful diagnostic tool in cancer cytogenetics (3-11). The high resolution of FISH analysis allows for a sensitive visualization of gene alterations. This has implications for the diagnosis of constitutional microdeletion syndromes (Fig. 1A), translocations in a variety of human diseases (12-18) as well as the identification of deletions of tumor suppressor genes and amplification of oncogenes in different types of human malignancies (19-25) (Fig. 1B). Fiber FISH technique allows to produce decondensed stretched interphase chromatin for orientation and ordering cosmids, PACs, BACs, and YACs while gener-

From: Methods in Molecular Medicine, vol. 50: Colorectal Cancer: Methods and Protocols Edited by: S. M. Powell © Humana Press Inc., Totowa, NJ

Fig. 1. Representative FISH data demonstrating: (A) Constitutional VHL gene deletion (chromosome 3p25) detected in VHL patient's blood lymphocytes using the cDNA probe g7 (rhodamine signal). A centromeric alpha-satellite probe specific for chromosome 3 (FITC signal) was used as a control. Chromosomes are counterstained with DAPI. (B) Allelic deletion of the MEN1 locus (chromosome 11q13) in pituitary adenoma tumor cells. Dual-color FISH was performed on tumor touch preparation

Fig. 1. Representative FISH data demonstrating: (A) Constitutional VHL gene deletion (chromosome 3p25) detected in VHL patient's blood lymphocytes using the cDNA probe g7 (rhodamine signal). A centromeric alpha-satellite probe specific for chromosome 3 (FITC signal) was used as a control. Chromosomes are counterstained with DAPI. (B) Allelic deletion of the MEN1 locus (chromosome 11q13) in pituitary adenoma tumor cells. Dual-color FISH was performed on tumor touch preparation ating contigs in particular region of interest, definition and approximate sizing of gaps and overlaps (Fig. 1C-E). These new high-resolution FISH technologies have widespread applications for long-range genome mapping.

Comparative Genomic Hybridization (CGH) is one of the applications of FISH. The method has been developed to detect an integral pattern of the genetic changes in the tumor genome including aneuploidies, extended chromosomal deletions (losses), presence of extra chromosomal fragments (gains) and amplifications (26-27) (Fig. 1F,G). The genomic DNA from tumor cells is the only requirement for the analysis. The principle of the method is the competitive hybridization of the tumor and referent normal DNAs labeled differently (used as a probe) to the metaphase chromosomes from the normal donor (used as a template). A specially designed software for CGH analysis allows to measure the ratio of hybridization efficiency tumor:normal DNA along the axis of each individual chromosome. This method is extremely valuable as the first approach to study genetically unknown cancer syndromes or unknown tumor entity, especially associated with the hereditary condition. By using CGH on a series of tumors it is possible to identify specific chromosomal rearrangements characteristic for this tumor type. Consistent chromosomal loss will indicate region of localization of the putative tumor-supressor genes. Consistent areas of gain or amplification would show location of the putative oncogenes involved in tumor initiation and progression. In one experiment it is possible to unravel all chromosomal losses and gains occurred in tumor genome.

In order to detect translocation another FISH technique can be applied, Spectral Karyotyping (SKY) (28). A combination of chromosomal painting probes labeled with different fluorochromes covering all 23 pairs of human chromosomes (or 20 pairs of the mouse chromosomes) is now commercially available using the MEN1 locus specific cosmid 10B11 (red signal) and chromosome-specific alpha-satellite for chromosome 11 (green FITC signal). FISH detects an aneusomy for chromosome 11. (C) Three-color FISH for high resolution mapping. Physical ordering and estimation of the distances between three BAC clones (orange-red-green) in the contig from Carney Complex critical region (chromosome 2p16) using depleted chromatin fibers as a template. (D, E) Fiber FISH using BAC clones from Carney Complex critical region, overlapped (D), and approx 80 kb apart from each other (E). (F, G) CGH analysis of squamous cell type esophageal carcinoma. (F) Normal metaphase spread after simultaneous hybridization of the tumor (FITC fluorescence) and normal referent (rhodamine fluorescence) DNAs. Chromosomal regions with loss appear red whereas areas of gain have extra green fluorescence. (D) The CGH image profile for the same tumor, computed as a mean value for ten metaphase spreads. The parallel vertical lines represent tumor:reference rations. The bold median line represents ratio of 1.0, and red and green line indicate ratio of 0.9 and 1.1, respectively. Red represents losses and green represents gains and amplification.

(Applied Spectral Imaging, Carlsbad, CA; Vysis, Inc., Downers Grove, IL). Requirement for this experiment is metaphase spreads obtained from the tumor cells.

How does FISH contribute to the study of neoplastic process and what are the main current advantages of the method?

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