Chrombios GmbH was founded in 1998 by scientists as a "spin off" of the University of Munich, Germany and the German Human Genome Project.
Based in the quickly growing biotechnology region around Munich, Bavaria, Germany, Chrombios is focused on service and products related to Molecular Cytogenetics.
More than 20 years of experience in Cytogenetics and Molecular Biology, a basket full of ideas and the confidence that Molecular Cytogenetics is an important component of genetics and pathology induced the two founders.
The company’s aims are to develop service and products for Molecular Cytogenetics and to speed up the exchange of ideas between universities and small business.
A very small overhead and effective research and development should keep our prices highly attractive. Various products have been developed in collaboration with colleagues in the field and Chrombios provides the expertise and infrastructure for a successful distribution of innovations.
Chrombios provides high quality Molecular Cytogenetic service and products with focus on the analysis of chromosomes in cancer and animal models in genome research. |
In particular, we offer for human and mouse:
Multi color FISH Service for human and mouse. This includes Color Karyotyping of cell lines (for example mouse ES cells), analysis of tumors, rearrangements after exposure to irradiation and to other mutagens,.....
Mapping of cloned DNA sequences on metaphase chromosomes.
Characterization of cell lines (for example for patent applications).
Comparative Genome Hybridization (CGH) of human and mouse tumors.
We can offer you:
Customized single or multi color probe sets (for example, pools of repetitive probes or chromosome paints in the same or in different colors).
In addition to human and mouse, Chrombios offers cytogenetic service on various other important animal models in genome research as well as for life stock species.
An Electronic Textbook on Basic Fluorescence in situ Hybridization (FISH) Techniques
Do you want to learn more about FISH technologies? In this electronic textbook we will cover the basic principles of FISH. This primer in FISH is intended for beginners, however, also advanced "FISHers" might find some useful hints.
FISH experiments can be performed with any DNA/RNA probe of sufficient size and incorporation of label to cell components that contain sequences complementary to the probe. The most widely used target, however, are metaphase chromosomes. Since chromosomes can be differentiated by their characteristics such as size, position of the centromere and banding pattern, hybridized probes can be localized on a highly defined genetic map. The resolution of this map is about one chromosome band that corresponds to a size of 5-10 Mega base pairs (Mbp).
When analyzing the order of different probes on the more relaxed DNA of interphase nuclei a higher resolution can be achieved. Probes that map less than 1 Mbp apart still give separate hybridization signals. An even higher resolution can be achieved when probes are hybridized on DNA fibers released from interphase nuclei by detergents or other techniques. DNA sequences of the size of a few Kilo base pairs (Kbp) can be mapped relative to each other on these extended chromatin fibers (fiber FISH). However, both interphase mapping and the use of chromatin fibers does not provide any information on the individual chromosome map.
Principle of indirect and direct labeling
As already introduced, Biotin-dUTP still serves as the most attractive non-radioactive label for FISH. Generally, for detection of the probe Avidin is used conjugated to a variety of different fluorochromes covering the entire visible spectrum of the light as well as some fluorochromes that emit in the near infrared. Since the probe is labeled with a hapten that itself is not fluorescent this procedure is called "indirect" detection (Figure 9). Other "indirect" labels include haptens like Dinitrophenol, Digoxigenin, or Oestradiol that can be detected by fluorochrome labeled antibodies. Again, antibodies against these haptens are now available that are coupled to numerous different fluorescent dyes.
"Direct" labels are dUTPs directly coupled to fluorochromes. After incorporation into the probe and following in situ hybridization the probe is visible without further need of detection procedures. Thus, a probe labeled for example with Cy3™ -dUTP (see below) is immediately visible after hybridization as a red fluorescence in the microscope (Figure 9). Again, a full range of "direct" labeled dUTPs are commercially available covering the entire visible spectrum of the light as well as the far red/close infrared. In many instances, the intensity of the fluorescence is not as strong as those provided by "indirect" systems. Since the workload in the "wet lab" to visualize the probe is much less compared to "indirect" labels and many FISH labs use electronic imaging of the signals, signal intensity may not be too important anymore. However, when analyzing very small probes "indirect" labels are still preferred.
Illustration of FISH with probes labeled indirectly with a hapten or directly with a fluorochrome. In FISH experiments with indirect labels (left side) such as Biotin or Digoxigenin the hapten itself is non-fluorescent. The probe is hybridized to the chromosomal DNA. After hybridization the signal becomes visible after application of a fluorescent antibody against the hapten or by fluorochrome labeled Avidin that binds to Biotin. FISH with direct labels uses fluorescent dUTPs incorporated in the probe (right side). With this technique there is no further need for detection of the hybridized probe after hybridization.
The fluorescence signal can be amplified by applying a "sandwich" technique of further fluorescent labeled antibodies. This can be used for direct as well as for indirect detection techniques. For example a fluorescent labeled antibody against Avidin can be used for signal amplification of the fluorescence emitted by the molecule bound to Avidine. Fluorescent labeled antibodies are also available that are directed against fluorochromes, thus amplifying the hybridization signal. Many of these systems, however, also amplify the "noise" of unspecific background fluorescence and in most instances a single "indirect" or "direct" label will give sufficient signal intensity.
Labeling by nick-translation or PCR
Hapten- or fluorescent labeled dUTPs are generally incorporated into the probe by either nick-translation or PCR. During nick-translation the double stranded probe is "nicked" by DNAse and partially digested. At the same time, the strand is filled up by a polymerase that incorporates dNTPs and also labeled dUTP offered in the labeling reaction. Nick-translation is usually used for labeling of cloned DNA such as BACs and YACs. However, PCR can also be used to lable these probes with degenerated primers (DOP-PCR). Usually DOP-PCR is used to generate and label complex probes such as paints from flow sorted chromosomes or micro-dissection. Most haptens and fluorochromes can withstand the high temperature cycles during PCR. PCR has the advantage that the entire probe is also being amplified. There are, however some haptens such as oestradiol-dUTP or some fluorochromes in the infrared that are not suitable for PCR labeling.
Counter staining chromosomes and nuclei
For FISH experiments, chromosomes and cell nuclei are generally counter stained by a fluorescent dye that is specific for DNA. The most common dye is DAPI (4´, 6-Diamidino-2-phenyl-indol) that is excited in the UV and chromosomes and cell nuclei show a bright blue fluorescence. Another common counter stain is propidium iodide that shows a red fluorescence. In experiments where several different probes should be visualized the best choice for a counter stain is DAPI.
Fluorochromes
The most frequent used dyes for the detection of the FISH signals are FITC (fluorescein isothiocyanate) that emits a green fluorescence and dyes with orange or red fluorescence, such as Cy3™ , or TexasRed™. Another commonly used fluorochrome in FISH experiments is Cy5™ that emits in the far red/close infrared. Since this fluorescence is not visible by eye it would need detection by an infrared sensitive camera mounted on the microscope. There are various other dyes with similar characteristics available from various different vendors. For example, FITC can well be replaced by Rhodamin 110, and Cy3™ by TAMRA (carboxytetramethyl- rhodamine) depending on the actual price and for some dyes, depending on the quality of the currently distributed lot.
The microscope setup: Overview
Basic setup for FISH microscopy
- The light source
- The fluorescence filter
- Fluorochromes and filters
Advanced setup and automation in FISH microscopy
- Automation of fluorescence microscopy
- Automation in karyotyping
A modern epifluorescence microscope should be able to display various fluorochromes as single dyes or in combinations. To set up a microscope for FISH the first thing to consider is the light source. Since the excitation of these fluorochromes needs a high energy light source usually high pressure mercury or xenon bulbs are used. Xenon bulbs (XBO) show a rather uniform light from the green to the infrared, however, mercury bulbs are better to use for dyes that are excited in the UV and blue light. The disadvantage of mercury bulbs is that they are weaker in the infrared. They also show various peaks and valleys in their spectrum. Thus, when using a fluorochrome that has an excitation maximum that falls directly into one of these valleys, the fluorescent signal may be disappointing. However, for most applications a 100W mercury bulb (HBO) is the optimal light source covering the excitation of most commonly used fluorescent dyes.
The fluorescence filters
The next thing to consider is the choice of the fluorescence filters that would allow an optimal excitation and detection of the emission of the dye as well as a clear differentiation between both excitation and emission. Typically, filters may allow light to pass beyond or above a certain wave length (short pass and long pass filters, respectively). Alternatively, they are designed to filter only a certain bandwidth of the light (band pass filters).
Modern filters for epi-fluorescence consist of three components: the excitation filter, emission filter and a beam splitter.The excitation filter is specifying the part from the spectrum of the light source that is optimal for the excitation of a given fluorochrome. The beam splitter is then directing the light to the specimen where the fluorochrome is excited to emit light in a longer wave length (stokes shift). Only the longer wave length light can pass the beam splitter. For a even better seperation of excitation and emission the fluorescent light is further differentiated by the emission filter (Figure 12, see also Figure 11 on previous page).
In the simplest configuration, for each fluorescent dye a single filter set is used. To allow the inspection of different fluorochromes these filters can be manually changed in a slider or, in a more advanced setup, moved in a motorized filter wheel. In case the hybridization signal should be analyzed directly in the microscope by eye the signal should be visible together with the counter stain. Thus, a filter should be used that fits both stains. For example, a simple filter set with a broad emission for both green and orange fluorescence will allow the visualization of both the FITC hybridization signal and the counter staining with propidium iodide. In this example the green hybridization signal will appear yellow on a red background staining of chromosomes. More complicated are multi band pass filters especially designed for specific fluorochrome combinations such as DAPI/FITC/Cy3™ or DAPI/FITC/ TexasRed™. Documentation of these fluorescent signals would need classical color photography, a color TV or a CCD camera.
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