In situ hybridization

In-situ hybridization ( ISH hybridization and in situ) is a molecular biological method to detect nucleic acids ( RNA or DNA) in tissues, individual cells or metaphase chromosomes. Here, a artificially prepared probe nucleic acid is used which binds through base pairing of the nucleic acid ( hybridization ). The proof is, not just as in the test tube (in vitro) performed, but directly in the respective structure ( in situ).

Prevalence have fluorescence in situ hybridization (FISH) for detection of DNA or RNA (RNA - FISH) in cell nuclei of individual cells or metaphase chromosomes, and the investigation of the distribution of mRNA in whole embryo sections or tissues with coloring ( chromogenic ) enzymes ( FISH test ).

Other names used are genomic in situ hybridization ( GISH ), is used in the gesamtgenomische as probe DNA, and chromosomal in situ Suppressionshybridisierung ( CISS hybridization), with the marker chromosomes of the whole possible.

• 4.1 Areas of application

History

In situ hybridization in the late 1960s was developed by the U.S. biologist Mary Lou Pardue and Joe Gall. They used radiolabeled probes ( tracers), the preparations then had to be placed on an X-ray film. In addition to the general problems associated with radioactivity, the spatial resolution was then compared with current techniques bad. Therefore, later translated by probes that were covalently attached to the marker molecules ( see below).

Operation

In-situ hybridization due to the pairing of complementary bases of two nucleic acid single strands. One of the two strands is thereby of a previously prepared and labeled probe and the other is present in the specimen and is to be detected. The first steps are therefore preparation of the preparation as well as preparation of the probe. The probe is usually composed of DNA, since it is more stable than RNA, and thus easier to handle in the laboratory. Also, DNA can replicate easier with today's methods. The labeling of the probe can be done indirectly with haptens (eg, digoxigenin, biotin, or 2,4-dinitrophenol ) or the FISH directly with fluorescent molecules such as FITC or Cy3. Commonly used methods for labeling the probes are nick translation and PCR.

Because DNA usually exists as a double strand, this must first be separated (also: melted or denatured ) are. Denaturation can be either by shifting the pH ( both acidic alkaline) or by heat. In a heat denaturation, the melting point is usually lowered by the addition of formamide, which weakens its strong polarity, the hydrogen bonds between the DNA single strands. May be achieved at temperatures of about 70-75 ° C, a melting already, whereby the structure of the chromosomes will be less badly damaged.

The actual hybridization takes, depending on the probe material and target sequence, from one hour to several days. Then lie in the preparation of hybrid molecules of the nucleic acids of the specimen and the probe. Unused or non-specifically bound probe molecules are washed and bound probe molecules can be detected. In the indirect labeling this is done via an antibody staining or in the case of biotin with avidin. The antibody (or avidin), in turn to fluorophores (for FISH ), or bound to the enzymes which form of dyes to be added chromogenic substrate. Both will then be evaluated microscopically.

Detection of mRNA with coloring enzymes

This application is mainly used digoxigenin- labeled RNA using. Digoxigenin may be detected using an antibody, which is coupled, for example with an enzyme. The enzyme, usually alkaline phosphatase or peroxidase, may be implemented by the addition of the reagents, a dye that is covalently bound in the tissue and therefore do not spread by diffusion. The detection of mRNA in tissues stained only those cells ( hybridized ) in which a gene to be examined is enabled are: Only here is the corresponding mRNA before. This is particularly used in developmental biology application. Here it is of particular interest to monitor the activity of a gene, for example during embryogenesis in situ. The embryonic or adult tissues must be fixed for staining at first; the activity can not therefore be tracked in real time, but is only a snapshot of the state, in which there was the fabric when it was fixed.

Expiration

The fabric to be dyed - for example, embryos of various model organisms such as Arabidopsis thaliana, Drosophila melanogaster, Xenopus laevis, Mus musculus, Danio rerio - is fixed using formaldehyde-containing solutions. It is then transferred into a buffer, and the labeled probe formamidhaltigen added. The hybridization time is dependent on the size of the embryo, and takes at least several hours. During this time, the probe can diffuse through the tissue and binds wherever there are complementary sequences in the mRNA. The following are some washing steps where excess, unbound probes are washed off and at the end of the dyeing, that can then be analyzed in detail using a microscope and documented.

In addition to total preparations, for example, fly embryos ( left figure ), the in situ hybridization can be performed on tissue - thin sections. In this way, larger specimens can be examined, such as human tissue or whole mouse embryos ( right panel).

Fluorescence in situ hybridization

In fluorescence in situ hybridization (FISH), the probe is detected using a fluorescent dye. This allows the simultaneous detection of multiple structures by using different fluorescent dyes, for example the detection of a chromosome with two genes lying thereon. Chromosome preparations and in cell nuclei up to seven different dyes have already been used successfully.

Nucleus of mouse fibroblasts. Fluorescence in situ hybridization, the territories of chromosomes 2 (red) and 9 ( green) were stained. DNA counterstaining in blue. ( Clicking on the image opens a panel with additional examples from other cell types of the mouse)

Genreiche and poor regions in human chromosomes. In metaphase chromosomes of a human female lymphocytes which Alu sequences were by fluorescence in situ hybridization labeled ( green), which are common particularly in gene-rich chromosome segments. For visibility genarmer regions, the DNA is colored red.

Areas of application

The possibility of relatively easy to determine the presence or the number of parts of the genome in the fluorescence microscope is used for clinical diagnosis in different disciplines.

For the detection of genetic diseases are used preparations of chromosomes in the metaphase typically prepared in the laboratory from proliferated cells from the patient or, in the prenatal diagnosis, of the cells of the fetus (see FISH test ). These probes are then hybridized to the chromosome, for example, 21 to test whether there are three instead of two, and thus there is present the cause of Down syndrome. Whether certain chromosomal regions are missing, such as the cat cry syndrome, can also be determined with probes for these regions. In principle, such studies can be carried out also in cell nuclei, so must be produced without metaphase. However, this has the disadvantage that not every core provides a correct result: If two random chromosomes are detected in the nucleus next to each other, then both can not recognize them as two signals. It must therefore be counted many cores and a comparison with known normal cores must be performed in order to make a reliable statement can. The advantage of this method lies in the higher speed, because a multiplication of the cells in the laboratory, or only to a lesser extent is necessary. Also in the characterization of marker chromosomes FISH is used.

In the examination of the genome of tumor cells FISH is used to determine the chromosome aberrations. For example, metaphase can be examined by FISH to detect chromosomal alterations, which can not be diagnosed by G- banding. This can be tested probes for various human chromosomes in order. Multicolor -FISH also allows to detect all chromosomes in different color combinations, so that all conversions between different chromosomes can be detected in an experiment .. The methods necessary for this purpose, however, are challenging and require special microscopic equipment. Some chromosomal alterations occurring in specific tumor types heaped upon, such as the Philadelphia chromosome in some leukemias. In non-Hodgkin's lymphomas different subgroups have different typical genetic changes. The presence of such changes can be targeted for testing with FISH.