In situ hybridization

The in situ hybridization is a method for detecting chromosomal aberrations. Certain chromosomes are marked with fluorescent dyes and bound to a DNA probe. This technique is used for prenatal diagnosis of gene mutations.

What is in situ hybridization?

In the case of in situ hybridization or the Fluorescence in situ hybridization nucleic acids from RNA or DNA are detected in certain tissues or a cell using molecular genetic methods. Usually this type of diagnosis is used to detect a structural or numerical chromosomal abnormality during pregnancy.

For this purpose, an artificially manufactured probe is used, which itself consists of nucleic acid. It then binds to the nucleic acids in the organism through base pairing. This bond is meant by the term hybridization. Evidence is based on the living structure of the patient and therefore corresponds to in-situ evidence. This is to be distinguished from in-vitro methods in which the detection takes place in the test tube. The method was developed in the 20th century by scientists Joe Gall and Mary Lou Pardue.

Technology has evolved since then. While radioactive probes were used at the time, for example, fluorescence-labeled probes with a covalent bond to the marker molecules are used today.

Function, effect & goals

In-situ hybridization is usually used to detect chromosomal aberrations, i.e. chromosomal anomalies that cannot be detected in a karyogram. This means that the method is always used when hereditary diseases are to be determined during pregnancy.

Since chromosome aberrations are a problem that should not be underestimated today, the use of the method has increased over time. Hybridization takes place on the basis of native cells from the mother's amniotic fluid. The basis of the technology is the binding of the color-marked probe to DNA parts. Thanks to the binding, the number of copies can later be evaluated with a microscope, since the individual copies emit a light signal and can thus be made visible under the microscope. There are different procedures. Either the analysis takes place immediately after binding.

In this case, a fluorescent dye such as biotin is used, which is bound directly to the DNA probe. With the indirect method of in-situ hybridization, the evaluation cannot take place immediately after the hybridization, since fluorescent substances can only bind to the probe after the hybridization. This indirect method is used more often than the direct one because it is believed to be more sensitive. In addition to chromosome-specific centrometer DNA probes, there are also locus-specific DNA probes, chromosome-specific DNA library probes and comparative genome hybridizations.

Chromosome-specific centrometer DNA probes can be used to detect chromosomally numerical anomalies. This means that they are mainly used when there is a suspicion of duplicated or deleted chromosomes. The location-specific DNA probes are particularly suitable for the detection of minimal mutations that cannot be detected in the karyogram. A chromosome-specific DNA library probe is used in particular to detect insertions and translocations.

Comparative genome hybridization, on the other hand, is a comprehensive analysis of losses and gains in chromosomal material. Today, in-situ hybridization is very important in the diagnosis of various chromosome mutations.In the diagnosis of Down syndrome, probes bind to chromosome 21, for example. For this purpose, chromosome-specific probes are usually used, which can be used if this disease is suspected.

A suspicion can arise, for example, if the parents have previously given birth to a child with the disease and the ultrasound image is abnormal. If there is a triple instead of a double bond and thus a triple color signal is output, the diagnosis is considered confirmed.

Risks, side effects & dangers

In contrast to, for example, PCR, in-situ hybridization is significantly less susceptible to contamination. In addition, the time required for the process is immensely less. However, because embryos in particular form chromosome patterns, the rest of the chromosomal distribution and thus the genetic status of other cells cannot be inferred from a given pattern.

Color signals can also overlap or remain invisible for other reasons. As a result, in-situ hybridization as a diagnostic tool is relatively prone to errors during pregnancy. Misdiagnoses can arise and parents may decide against a healthy embryo. In order to reduce the susceptibility to errors of in situ hybridization, at least two embryonic cells should be examined simultaneously. By examining two cells in parallel, there is now only a negligible risk of misdiagnosis.

In such a case, parents can absolutely rely on the diagnosis. In-situ hybridization is not offered to every pregnant woman, but only to women from a risk group. Nevertheless, pregnant women are not denied this type of diagnosis at their own request. Abnormal ultrasound findings or an abnormal serum can induce a doctor to offer the diagnostic procedure. Although a large part, but by no means all, of chromosomal aberrations can be diagnosed using in situ hybridization.

Therefore, the in-situ hybridization must never be carried out alone, but must always be used in conjunction with a conventional chromosome test. The care of the pregnant woman plays a major role in this procedure. Before the analysis, there is an in-depth discussion with the expectant mother about the diagnostic method, which explains the risks, the possibilities and the limits of the technology.