Chromogenic in situ hybridization
Related Terms
Amplification, cancer, chromosome, CISH, colcemid, cytogenetic technique, deletion, disease, DNA, karyotype, microscope, probe.
Background
Chromogenic in situ hybridization (CISH) is a method that researchers may use to determine whether a specific part of a chromosome is present in a cell. Chromosomes are located in a compartment of the cell called the nucleus and are composed of deoxyribonucleic acid (DNA) and proteins. Human cells contain 46 chromosomes, 22 pairs of autosomes and one pair of sex chromosomes, and each chromosome has hundreds of genes. Genes contain the instructions for making the proteins that perform all of the functions in the human body, such as producing energy.
To perform CISH, researchers may first identify a specific region to study, for example, a region that contains a certain gene of interest. They then generate a probe, which is a sequence of DNA that can recognize and bind to the chromosomal region of interest. In CISH, the probes are designed to generate a colored stain, so that if a chromosomal region of interest is present in a cell, researchers will be able to see the stain by looking at a cellular sample under the microscope.
In some diseases, such as cancer, genetic mutations occur that can cause part of a DNA sequence in the chromosome to be deleted, repeated, or reversed in orientation. By looking for differences in how chromosomes from different cells stain with a probe, researchers may be able to observe these changes under the microscope and learn more about the genetic mutations that cause a particular disease.
Methods
Obtain cells: To perform chromogenic in situ hybridization (CISH), a line of cells that a researcher is interested in studying is first grown in the laboratory. Cells lines made from blood, amniotic fluid, or bone marrow are commonly used for analysis. After the cells have multiplied, a chemical, such as colcemid, is used to stop the cells from growing further. Colcemid inhibits the growth of cells when they reach a stage of the cell cycle called metaphase, when the chromosomes are easily visible. Colcemid stops the cells from growing by interfering with the function of microtubules, which are proteins needed by the cell for growth.
Prepare cells for observation: Once the cells have been obtained, the cells are exposed to a hypotonic solution, that is, a solution with a relatively low concentration of dissolved molecules. This solution expands the cells and spreads the chromosomes out so they will be easier to examine. The cells are killed using a preserving agent called a fixative, such as methanol and acetic acid, and then transferred to a microscope where the chromosomes can be observed. Some of the metaphase cells are dropped onto a microscope slide (a thin piece of glass) and then spread apart on the slide with a small tip. They are then dried in the air so they become attached to a cell.
Obtain probe: To perform CISH, researchers may first identify a region of a chromosome they wish to study, for example, a region that contains a specific gene. They then generate a probe, which is a sequence of DNA that can recognize and bind to the chromosomal region of interest. Probes can recognize the DNA because the bases in the probe bind to the bases in the DNA sample. Researchers can obtain the DNA material for a probe in several different ways. For example, they may perform a technique called chromosome microdissection, in which they physically remove part of a chromosome and then use it as a probe. Or they may obtain a piece of DNA to use as a probe from a DNA library, which is a large collection of isolated and purified DNA fragments that is frequently used in DNA sequencing projects.
Apply probe to cells: Using the probe, researchers can determine whether the cells they are studying contain the region of DNA that the probe was designed to detect. The cells, which have been attached to microscope slides, are heated so that the DNA will be more accessible to the probe. The probe is then added to the slide. If the chromosomal region of interest is present, the probe will bind to the chromosome in that region. After the probe has been given time to bind to the DNA, a process that may take up to several days, the cells are washed so that probe material remains only in cells where it has detected and bound to a chromosomal region.
In CISH, the probes that researchers use are designed to generate a colored stain, so that if a chromosomal region of interest is present in a cell, researchers will be able to see the stain by looking at a cellular sample under the microscope. To generate the colored stain, an enzyme, a type of protein that helps carry out chemical reactions, is linked to the probe. When a certain type of chemical is then added to the cellular sample, the enzyme carries out a reaction on the chemical, causing a colored stain to form. These colored stains can be observed by researchers using a standard light microscope. An enzyme called peroxidase and a chemical called diaminobenzidine are commonly used in CISH, which cause a dark brown color to form.
Research
Using chromogenic in situ hybridization (CISH), researchers can check for differences in the chromosomes between two different cells. In some cases, CISH may be able to demonstrate that one cell has an extra copy of a specific chromosome. In other cases, CISH may be able to detect smaller variations in a particular chromosome, such as a repeated region or a deletion in the chromosome. CISH is often used to study different types of diseases, such as cancer, in which mutations occur that cause extra copies of chromosomes to be present, or that cause a portion of a chromosome to become deleted, repeated, or reversed in orientation.
CISH may be used to study evolutionary relationships between different species. If a probe is designed to detect a specific chromosomal region in one species, that probe can also be used to check for the same chromosomal region in another species. This may let researchers know whether a specific gene is present in both species. If the species are closely related, the probe will also bind to the chromosome of the other species. If the species are distantly related, however, the probe will no longer be able to bind to the chromosome of the second species because the DNA sequences are too different. For example, humans and yeast are distantly related organisms, so CISH probes for some human genes may not be able to detect a similar gene in yeast. CISH may also be used to look for other changes that occur between two species during evolution, such as increased copy numbers of a particular chromosomal region.
Implications
Diagnosis: Chromogenic in situ hybridization (CISH) can be used to diagnose certain human diseases in a developing fetus. For example, genetic diseases such as trisomy 18 or Down syndrome (also known as trisomy 21) may be diagnosed in a developing fetus through amniocentesis, in which the amniotic fluid surrounding the fetus is sampled through a needle. Cells in the amniotic fluid can be used to perform CISH and check for an extra copy of chromosome 18 in the case of trisomy 18, or an extra copy of chromosome 21 in the case of Down syndrome. However, performing prenatal testing does carry a risk to the fetus. And in many genetic diseases, there may be no way to prevent the disease from developing once it has been diagnosed.
Study cancer: CISH may be used by doctors to study some types of cancer. Using CISH, researchers have found that in lung and brain cancer cells, a specific gene called the epidermal growth factor receptor is present in more copies than in normal cells. Genetic changes have been observed using CISH in other types of cancer as well, such as breast and stomach cancer. These genetic changes may be used by doctors to diagnose cancers or to determine the progression of the disease.
Understanding disease: By understanding the specific rearrangements that chromosomes have undergone in patients, CISH can help researchers better understand some diseases. This is because rearrangements may cause specific genes to become deleted or amplified and the genes that have decreased or increased in number may play a role in causing the disease. By identifying these genes, scientists can better understand how the disease is caused, and they may be able to use this information to develop drugs to fight the disease. This approach is useful for the study of some types of cancer, in which chromosomes become mutated and undergo rearrangements.
Limitations
One drawback of chromogenic in situ hybridization (CISH) is that in order to generate a probe to detect a specific region of a chromosome, some information about the DNA sequence of that chromosome must already be known. It is not always necessary to know the specific DNA sequence of the area that is being examined, and researchers sometimes generate probes based on regions of chromosomes near the regions they are interested in studying.
Researchers must also be careful when generating a probe. Some regions on a specific chromosome may be very similar to regions on a different chromosome, so the probe may be able to bind to another chromosomal region. Researchers therefore may need to generate a probe that is specific only to the region they are interested in but that is not similar to other regions.
Future research
Not applicable.
Author information
This information has been edited and peer-reviewed by contributors to the Natural Standard Research Collaboration (www.naturalstandard.com).
Bibliography
Di Palma S, Collins N, Bilous M, et al. A quality assurance exercise to evaluate the accuracy and reproducibility of chromogenic in situ hybridisation for HER2 analysis in breast cancer. J Clin Pathol. 2008 Jun;61(6):757-60.
National Human Genome Research Institute (NHGRI). .
Natural Standard: The Authority on Integrative Medicine. .
Park DI, Yun JW, Park JH, et al. HER-2/neu amplification is an independent prognostic factor in gastric cancer. Dig Dis Sci. 2006 Aug;51(8):1371-9.
Quezado M, Ronchetti R, Rapkiewicz A, et al. Validation Chromogenic in situ hybridization accurately identifies EGFR amplification in small cell glioblastoma multiforme, a common subtype of primary GBM. Clin Neuropathol. 2005 Jul-Aug;24(4):163-9.
Sholl LM, John Iafrate A, Chou YP, et al. Validation of chromogenic in situ hybridization for detection of EGFR copy number amplification in nonsmall cell lung carcinoma. Mod Pathol. 2007 Oct;20(10):1028-35.
Todorovic-Rakovic N, Jovanovic D, Neskovic-Konstantinovic Z, et al. Prognostic value of HER2 gene amplification detected by chromogenic in situ hybridization (CISH) in metastatic breast cancer. Exp Mol Pathol. 2007 Jun;82(3):262-8.