In Situ Hybridization

In situ hybridization is a technique used to detect specific DNA and RNA sequences in a biological sample. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are macromolecules made up of different sequences of four nucleotide bases (adenine, guanine, uracil, cytosine, and thymidine). In situ hybridization takes advantage of the fact that each nucleotide base binds with a complementary nucleotide base. For instance, adenine binds with thymidine (in DNA) or uracil (in RNA) using hydrogen bonding . Similarly, guanine binds with cytosine.

In a specialized molecular biology laboratory, researchers can make a sequence of nucleotide bases that is complementary to a target sequence that occurs naturally in a cell (in a gene, for example). When this complementary sequence is exposed to the cell, it will bind with that naturally occuring target DNA or RNA in that cell, thus forming what is known as a hybrid. The complementary sequence thus can be used as a "probe" for cellular RNA or DNA.

Thus, the term "hybridization" refers to the chemical reaction between the probe and the DNA or RNA to be detected. If hybridization is performed on actual tissue sections, cells, or isolated chromosomes in order to detect the site where the DNA or RNA is located, it is said to be done "in situ." By contrast, "in vitro" hybridization takes place in a test tube or other apparatus, and is used to isolate DNA or RNA, or to determine sequence similarity of two nucleotide segments.

Application of the Probe for DNA or RNA to Tissues or Cells

In situ hybridization allows us to learn more about the geographical location of, for example, the messenger RNA (mRNA) in a cell or tissue. It can also tell us where a gene is located on a chromosome. Obviously, a detection system must be built into the technique to allow the cytochemist to visualize and map the geography of these molecules in the cells in question.

When in situ hybridization was first introduced, it was applied to isolated cell nuclei to detect specific DNA sequences. Early users applied the techniques to isolated chromosomal preparations in order to map the location of genes in those chromosomes. The technique has also been used to detect viral DNA in an infected cell. In situ hybridization of RNA has also been used to show that RNA synthesis (transcription) occurs in the nucleus, while protein synthesis (translation) occurs in the cytoplasm .

Conditions that Promote Optimal In Situ Hybridization

Hybrid probes are known as cDNA or cRNA, because they are complementary to the target molecule. In developing an in situ hybridization protocol , it is vital to learn optimal temperatures and times needed for formation of the hybrid between the cDNA probe or cRNA probe and unique RNA or DNA in the cell. The optimal hybridization temperature depends on several factors, including the types of bases in the target sequence and the concentration of certain ingredients in the media. The concentration of cytosine and guanine in the sequence plays an important role. A cytochemist will use these factors to calculate optimal temperature when planning the experiment. One must be as careful in setting up an in situ hybridization experiment as one is when setting up a test tube hybridization assay. The cytochemist and molecular biologist work together to optimize the conditions.

Another vital consideration in developing good in situ hybridization techniques is the specificity of the probe itself. If the investigators know the exact nucleotide sequence of the mRNA or DNA in the cell, they can design a complementary probe and have it made in a molecular biology lab. However, if the investigators do not know the exact sequence, they may try a sequence that is as close to exact as possible (such as from a related species). For example, they might try a cDNA probe for a mouse DNA sequence on a tissue preparation from a rat. This may or may not work because, if over 5 percent of the base pairs are not complementary, the probe will bind only loosely to the target. This loose binding may cause it to be dislodged in the washing or detection steps and hence the reaction will not be detected, or only some of the sites may be detected and the labeling will not accurately reflect all of the target sites.

Application of Tools to Detect the Hybridization Sites

Forming the hybrid is not sufficient to allow it to be mapped, because the molecules are too small to be seen in the microscope. Thus, to map the geographical distribution of the gene or gene product, the cytochemist applies sensitive detection systems that allow the hybrid to be seen. This is done by labeling the cDNA or cRNA probe itself with a molecule that can either be visualized directly (such as fluorescein, which glows when exposed to fluorescent light in a microscope) or indirectly (such as radioactive sulfur, which can be detected by autoradiography; or biotin, which can be detected by avidin or by specific antibodies to biotin). Whichever type of molecule is chosen, it should be small, so that it does not interfere with the hybridization process.

The molecule used to visualize the hybrid is called the reporter molecule because it "reports" the site of the hybridization of the probe to the cellular DNA or RNA. Many different detection systems are available to cytochemists, employing a wide variety of reporter molecules. These include fluorescent compounds, colloidal gold compounds, or enzyme reactions or radioactive elements. Cytochemists will choose the most sensitive detection system that is also appropriate for their laboratories. For example, laboratories that do not want to work with radioactive compounds may choose one of the many nonradioactive methods.

One of the best-known nonradioactive in situ hybridization methods is FISH or "fluorescence in situ hybridization." It allows the detection of many genes in a chromosome or a nucleus, and different combinations of fluorescent reporter molecules are used to produce different colors. Using FISH, a veritable rainbow of colors can be used to map the location of genes on chromosomes. Another method uses enzyme reactions to form a product over the site of the mRNA or DNA in the cell. One can use different enzymes or enzyme substrates in the detection system and thus detect multiple gene products in the same tissue or cells.

Preserving the Tissues or Cells and Preventing Loss of DNA or RNA

Another important consideration in developing in situ hybridization technology involves the preservation of the cells or chromosomes. It is important to preserve the morphology (shape) and geographical site in the cell or chromosome where the target DNA or mRNA is located. Investigators may choose to use frozen sections, or they may treat cells or tissues with fixatives that cross-link proteins and stabilize cell structure. This prevents destruction during the hybridization and washing protocols.

Preserving DNA is easy because it is a highly stable molecule. However, preserving RNA is much more difficult because of a very stable enzyme called RNase, which may be found on glassware, in lab solutions, or on the hands of the cytochemist. RNase will quickly destroy any RNA in the cell or the RNA probe itself. Thus, investigators that work with RNA must use sterile techniques, gloves, and solutions to prevent RNase from contaminating and destroying the probe or tissue RNA.

Controlling the Specificity of the Cytochemical Assays

Good cytochemists know that experimental results must be checked and verified. For this reason, in addition to testing for reactions with their target DNA or RNA, they also test for reactions with unrelated nucleotide sequences. Likewise, they test for reactions with other components of the detection systems.

There are a series of controls that are run that detect if the labeling pattern is due to the proper sequence of reactants. For example, if the cyto-chemist leaves out the probe in the hybridization solution, there should be no reaction. Similarly, if the cytochemist changes the sequence of the probe, or uses a noncomplementary probe, there should be no reaction (unless that new sequences reacts with another sequence in the cell). Tests of the detection system must also be run by leaving out one or more components to learn if the reaction is dependent totally on the complete sequence of reactants.

see also DNA; Nucleotide; RNA.

Gwen V. Childs

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