FISH - Fluorescent In-situ Hybridization

FISH uses fluorescently labeled single-stranded DNA probes to bind complementary sequences on chromosomes within fixed cells. The probe hybridizes to its target DNA, allowing direct visualization of specific genes under a fluorescent microscope. This technique enables detection of gene locations and chromosomal abnormalities in cell-based diagnostic assays.

Cells are first arrested in interphase or metaphase, then suspended in buffer solution and spread on a clean glass slide. Next, both target DNA and labeled probes are denatured using heat or chemicals to separate double-stranded DNA into single strands capable of base pairing. This preparation allows the probe and target sequences to hybridize effectively.

The denatured probe and target DNA are mixed together and incubated overnight, allowing the probe to bind to its complementary sequence on the chromosomes. After hybridization is complete, excess probe is washed away. If the probe contains a fluorophore, the hybridized DNA can be directly visualized with a fluorescent microscope.

When probes lack direct fluorophore labels, they contain a modified nucleotide bonded to a hapten—a small molecule to which fluorescent antibodies or other fluorophores can attach. This indirect labeling approach allows fluorescent visualization of hybridized DNA through the attachment of labeling dna probes radioisotopes fluorophores biotin digoxigenin to the hapten.

FISH identifies chromosomal aberrations including aneuploidy (abnormal chromosome numbers), microdeletion and microduplication syndromes (loss or gain of chromosome segments), and subtelomeric rearrangements. High-resolution FISH images help detect these genetic abnormalities from loss, gain, or rearrangement of chromosomes during cell division.

Short fluorescence-labeled DNA probes bind to microbial 16s rRNA sequences in human blood cells, enabling visualization under a fluorescence microscope. This approach rapidly identifies pathogens at the genus or species level, particularly useful for slow-growing organisms like Mycobacteria that cause tuberculosis or difficult-to-culture microbes.

Advances in optical techniques, CCD cameras, and sophisticated image-processing software have enhanced FISH signal interpretation with greater sensitivity and specificity. These improvements have expanded FISH applications from cytogenetics and gene mapping to diagnosis of genetic and infectious diseases, enabling detection of chromosomal aberrations with high precision.