FISH | FISH

Key Words: CGH, cytogenetics, Fluorescence, fluorescence filters, fluorophore, mFISH

Definition:A cytogenetic method of detecting and localizing specific DNA sequences on chromosomes

TECHNOLOGY:

FISH is based on the principle that single stranded DNA will recognize and bind to its complementary sequence on a metaphase chromosome or within an interphase nucleus. It is usually carried out on standard cytogenetic preparations on microscope slides, but may also be carried out on other nuclear preparations, fixed cells and tissues, and blood and bone marrow smears.

The DNA is first denatured. Both target DNA and a complementary DNA (cDNA) probe, specific to the target nucleic acid sequence, are labeled with fluorescent molecules. Direct labeling, in which the fluorophore is bound directly to the probe allows examination of the sample directly after wash steps (to remove excess probe) and hybridization. Indirect labeling involves attachment of haptens to the probes before hybridization, which are then conjugated to fluorescently tagged antibodies, to enhance fluorescence signal, following hybridization.

Specimens are examined using fluorescence microscopy using filters appropriate to the probes used. FISH may identify entire chromosomes, chromosomal-specific regions, or single-copy unique sequences, depending on the labeling techniques used, the sensitivity of the system and whether amplification techniques have been used (PCR). FISH enables detection of morphological abnormalities and the localization of specific genes. While images can be analyzed manually, it is common to employ image analysis software in combination with automated microscopy to increase efficiency and accuracy.

APPLICATIONS:

FISH is widely used in genome studies, cytogenetics, cancer studies, biomedical research, microbiology, clinical diagnosis and prenatal diagnosis.

MICROSCOPE CONFIGURATION:

FISH can be carried out on any clinical / research level microscope capable of fluorescence imaging such as the Eclipse i-series. Automated systems (hardware and software) are often used.

SYSTEM SOLUTION:

Nikon's automated / manual Eclipse i-series clinical / research microscopes with epi-fluorescence capability. The Eclipse 90i is ideally suited to the capture of low light fluorescence data and enables automated image capture. A variety of commercially available cytogenetic data analysis software packages are available to facilitate image interpretation.

LINKS:

microscopyu.com/articles/fluorescence/insitu/brennerinsitu