Fluorescence in situ hybridization

It was developed by biomedical researchers in the early 1980s[1] to detect and localize the presence or absence of specific DNA sequences on chromosomes.

FISH is often used for finding specific features in DNA for use in genetic counseling, medicine, and species identification.

In biology, a probe is a single strand of DNA or RNA that is complementary to a nucleotide sequence of interest.

FISH is used by examining the cellular reproduction cycle, specifically interphase of the nuclei for any chromosomal abnormalities.

Probes are often derived from fragments of DNA that were isolated, purified, and amplified for use in the Human Genome Project.

The purpose of using RNA FISH is to detect target mRNA transcripts in cells, tissue sections, or even whole-mounts.

[10] The process is done in 3 main procedures: tissue preparation (pre-hybridization), hybridization, and washing (post-hybridization).

Some commonly used fixatives are 4% formaldehyde or paraformaldehyde (PFA) in phosphate buffered saline (PBS).

These steps aim to remove nonspecific hybrids and get rid of unbound probe molecules from the samples to reduce any background signaling.

The results are then visualized and quantified using a microscope that is capable of exciting the dye and recording images.

The differences between the various FISH techniques are usually due to variations in the sequence and labeling of the probes; and how they are used in combination.

In the opposite situation—where the absence of the secondary color is pathological—is illustrated by an assay used to investigate translocations where only one of the breakpoints is known or constant.

Targets can be reliably imaged through the application of multiple short singly labeled oligonucleotide probes.

Probes not binding to the intended sequence do not achieve sufficient localized fluorescence to be distinguished from background.

The technology has potential applications in cancer diagnosis,[19] neuroscience, gene expression analysis,[20] and companion diagnostics.

The preparation of fiber FISH samples, although conceptually simple, is a rather skilled art, and only specialized laboratories use the technique routinely.

[21] Q-FISH combines FISH with PNAs and computer software to quantify fluorescence intensity.

Flow-FISH uses flow cytometry to perform FISH automatically using per-cell fluorescence measurements.

Three primary fluorophores are able to generate a total of 7 readily detectable emission spectra as a result of combinatorial labeling using DOT.

The technology offers faster scoring with efficient probesets that can be readily detected with traditional fluorescent microscopes.

It uses combinatorial labeling, followed by imaging, and then error-resistant encoding[25] to capture a high number of RNA molecules and spatial localization within the cell.

The software, created for all scientists, not just bioinformaticians, reads a set of images, removes noise, and identifies RNA molecules.

In cases where the child's developmental disability is not understood, the cause of it can potentially be determined using FISH and cytogenetic techniques.

FISH, on the other hand, does not require living cells and can be quantified automatically, a computer counts the fluorescent dots present.

However, a trained technologist is required to distinguish subtle differences in banding patterns on bent and twisted metaphase chromosomes.

This technology is still in a developmental stage but, like other lab on a chip methods, it may lead to more portable diagnostic techniques.

[28][29] FISH has been extensively studied as a diagnostic technique for the identification of pathogens in the field of medical microbiology.

[31] Virtual karyotyping is another cost-effective, clinically available alternative to FISH panels using thousands to millions of probes on a single array to detect copy number changes, genome-wide, at unprecedented resolution.

Spectral karyotyping involves FISH using multiple forms of many types of probes with the result to see each chromosome labeled through its metaphase stage.