Fluorescence in situ hybridization

Lore & Background

FISH was developed in the early 1980s by biomedical researchers to detect and localize specific DNA sequences on chromosomes. The technique relies on fluorescent probes—single strands of DNA or RNA complementary to a target sequence—that bind to chromosomes or RNA targets. Fluorescence microscopy then reveals where the probe has bound. Probes are often derived from DNA fragments isolated and amplified during the Human Genome Project, with fragments around 100,000 base pairs stored in bacterial artificial chromosomes (BACs) in libraries such as the RPCI-11 library at Roswell Park Comprehensive Cancer Center.

The preparation and hybridization process differs for RNA and DNA targets. For RNA FISH, tissue samples are fixed with formaldehyde or paraformaldehyde, permeabilized with detergents like Triton X-100, and hybridized with probe pairs of 20–50 oligonucleotides. Signal amplification through sequential hybridization steps allows multiplexing of up to two targets. For DNA FISH, probes are tagged with fluorophores or biotin, applied to chromosome preparations, and incubated for about 12 hours. Wash steps remove unbound probes, and weak signals may be amplified using fluorescently tagged antibodies or streptavidin.

Variations in FISH techniques arise from differences in probe sequence, labeling, and combination. Probes can be designed to paint whole chromosomes, detect specific loci, or identify centromeric regions. The overlap of probes defines the resolution for detecting features like translocation breakpoints. FISH is used to examine interphase nuclei for chromosomal abnormalities and to analyze archival cases.

Reader's Guide

Fluorescence in situ hybridization (FISH) is a foundational technique in molecular cytogenetics, enabling the precise localization of DNA and RNA sequences within cells and on chromosomes. Its significance lies in its ability to bridge molecular biology and cytogenetics, providing visual evidence of genetic abnormalities and gene expression patterns. FISH is routinely applied in genetic counseling to detect chromosomal anomalies, in medicine for cancer diagnostics (e.g., identifying translocations or deletions), and in species identification. The technique's versatility extends to RNA targets, allowing researchers to map the spatial and temporal expression of mRNA, lncRNA, and miRNA in tissues and circulating tumor cells. By using probes derived from the Human Genome Project's BAC libraries, FISH offers high specificity and resolution. Its legacy includes enabling large-scale archival studies and multiplex assays, though signal strength and probe design remain critical factors. FISH remains a standard tool for both clinical diagnostics and basic research, particularly in understanding chromosomal structure and gene regulation.

Did You Know?

• Probes for FISH are often derived from DNA fragments of about 100,000 base pairs stored in bacterial artificial chromosomes.
• RNA FISH uses probes composed of 20–50 oligonucleotide pairs, each pair covering 40–50 base pairs.
• FISH can detect specific RNA targets including mRNA, lncRNA, and miRNA in cells and tissue samples.