Fluorescent in situ hybridization

From Wikipedia, the free encyclopedia

A metaphase cell positive for the bcr/abl rearrangement using FISH

FISH (Fluorescent in situ hybridization) is a cytogenetic technique which can be used to detect and localize the presence or absence of specific DNA sequences on chromosomes. It uses fluorescent probes which bind only to those parts of the chromosome with which they show a high degree of sequence similarity. Fluorescence microscopy can be used to find out where the fluorescent probe bound to the chromosome.

1 Process
- 1.1 Interphase FISH
- 1.2 Fibre FISH
2 Applications
3 See also
4 External links

[edit] Process

Image:FISH (technique).gif

First, a probe is constructed. The probe has to be long enough to hybridize specifically to its target (and not to similar sequences in the genome), but not too large to impede the hybridization process, and it should be tagged directly with fluorophores, with targets for antibodies or with biotin. This can be done in various ways, for example nick translation and PCR using tagged nucleotides.

Then, a chromosome preparation is produced. The chromosomes are firmly attached to a substrate, usually glass. After preparation the probe is applied to the chromosome DNA and starts to hybridize. In several wash steps all unhybridized or partially hybridized probes are washed away. If signal amplification is necessary to exceed the detection threshold of the microscope (which depends on many factors such as probe labeling efficiency, the kind of probe and the fluorescent dye), fluorescent tagged antibodies or streptavidin are bound to the tag molecules, thus amplifying the fluorescence.

Finally, the sample is embedded in an anti-bleaching agent and observed on a fluorescence microscope.

[edit] Interphase FISH

Interphase cells positive for a t(9;22) rearrangement

In interphase FISH the probe is applied to preparations with intact nuclei, either cytospins, paraffin sections, or even nuclei extracted from paraffin blocks. Hybridization is carried out similarly. The fluorescent signals are seen as dots in the cell nucleus, which is usually counterstained with a dye that recognizes DNA.

[edit] Fibre FISH

In fibre FISH, interphase chromosomes are attached to a slide in such a way that they are stretched out in a straight line, rather than being tightly coiled, as in conventional FISH, or adopting a random conformation, as in interphase FISH. This is accomplished by applying mechanical shear along the length of the slide; either to cells which have been fixed to the slide and then lysed, or to a solution of purified DNA. The extended conformation of the chromosomes allows dramatically higher resolution - even down to a few kilobases. The preparation of fibre FISH samples, although conceptually simple, is a rather skilled art, meaning only specialised laboratories use it routinely.

[edit] Applications

FISH can be used to map sequences to a specific position on a chromosome. While there are other ways to do this, the main advantage of FISH is that it is not dependent on recombination and thus can be used in chromosome regions where recombination is suppressed, such as the centromere. It can be used to map repetitive sequences that occur at several places on a chromosome.

FISH can also be used to do chromosome painting to make a comparison between two species or varieties by using DNA from entire chromosomes or even the entire genome of one species/variety as a probe on the other. In this way, Chromosomal abnormalities can be identified and evolutionary relations can be deduced.

FISH can be used to identify microorganisms, and is widely used in the field of microbial ecology. Biofilms for example are complex (often) multispecies bacterial organisations. Preparing DNA probes for one species and performing FISH with this probe allows one to visualize the distribution of this specific species in the biofilm. Preparing probes (in two different colors) for two species allows to visualize/study co-localization of these two species in the biofilm, revealing the fine architecture of the biofilm.

FISH has applications in clinical studies. To detect if a patient is infected with a suspected pathogen, bacteria sampled from the patient's tissues or fluids is typically grown on agar to determine the identity of the pathogen. Many bacteria however, even well known ones, don't grow well under laboratory conditions. FISH can be used to dectect directly the presence of the suspect on small samples of patient's tissue.

Bacterial FISH probes are often primers for the 16s rRNA region.