1. Principle of the CCK procedure
Multicolor karyotyping procedures, such as multiplex FISH (M-FISH), spectral karyotyping (SKY) or color changing karyotyping (CCK) allow identification of all chromosomes in a metaphase preparation (46 chromosomes in humans) based on color differences. They are possible because of the combinatorial labeling procedure (click here to review the principle of combinatorial labeling).
An alternative multicolor karyotyping technique, called color-changing karyotyping (CCK), has been described (Nature Genetics 23 (3): 263-4). This procedure allows simultaneous hybridization and separate detection of up to 41 different DNA probes using only three fluorescent dyes, making it appropriate for multicolor karyotyping. This makes it possible to karyotype all human chromosome based on color differences using any conventional epifluorescence microscope equipped with only three filters. Image capturing can be done using any cooled, non-cooled CCD or even a simple digital photographic camera. Specialized software is not required for image analysis, which can be performed with generic image processing software (Adobe Photoshop), on any computer platform. This procedure should make karyotyping by FISH affordable to any laboratory, as it does not require additional investments in cameras, filters, software and other equipment.
As in M-FISH/SKY, there is only one hybridization step, but the same metaphase has to be imaged twice: first image will reveal only the fluorescent-labeled chromosomes, whereas the second image will be taken after the coverslip is removed and fluorescent antibodies added to detect the haptene-labeled chromosomes (haptene = biotin, digoxigenin, dinitrophenyl).
Principle of the CCK procedure
DNA painting probes labeled with a combination of commercially available fluorescent- and haptene- labeled nucleotides are simultaneously hybridized on slides, but are visualized/imaged in a two-step process (see below). This "color-changing FISH" approach (Fig. 1 & 2) is possible because the strength of the fluorescent signal emitted by the labeled antibodies used to detect the three haptenes greatly overpowers the fluorescence emitted by the fluor-labeled nucleotides. Imaging of fluor-labeled antibodies requires 5-10x less exposure times, thus rendering the signal from the fluorescent nucleotides insignificant.
Fig 1 legend.Example of a "3+3" CCK labeling combination. This combination shown is for teaching purposes only and does not match the actual combination (Table 1) used in the experiment. Chromosomes are labeled with combinations of fluor-dUTP (colored circles) and haptene-dUTP (colored squares). The haptenes themselves do not fluoresce. Thus, after hybridization, imaging of a metaphase reveals only chromosomes labeled with dye-dUTPs ("direct" detection step). After imaging, the coverslip is removed and fluorescent antibodies applied to the slide to detect the haptenes ("indirect" detection step). Then, the same metaphases are imaged again. Because the fluorescence signal from labeled antibodies is much stronger, it overpowers the initial signal coming from the fluor-dUTP, thus shifting or "changing" the color of many chromosomes.
Fig. 2. Comparison between a "2+3" CCK and M-FISH.Both procedures use five different modified-nucleotides (dUTP conjugated with the fluors FITC, Cy3 or with the haptenes BIO, DIG, DNP). To simplify the discussion, image 2 depicts "cells" containing only 5 different chromosomes, each of them being differently labeled.In M-FISH, antibodies stained with three new dyes (for example Cy3.5, Cy5 and Cy5.5) are used to detect the three haptenes. The five fluors are sufficient to differentiate the chromosome by color. In a "2+3" CCK, the initial hybridization is visualized first. Only the fluor-labeled chromosomes are visible and some chromosomes have the same color or no color, as the haptenes themselves are not fluorescing. Then the coverslip is removed, the haptenes detected with fluorescent antibodies and the same metaphases are imaged again. The antibodies are labeled with the same fluors (FITC, Cy3) plus a third one (AMCA). Thus, although in the first image, the chromosome labeled with FITC and the one labeled with FITC+DIG appear to have the same color (green), in the second image (after adding the Cy3-labeled anti-DIG antibody), the chromosome labeled with DIG-dUTP changes (shifts) its color from green to red. Because the red color from the antibody is very bright, the chromosome only shows its red color. The previously green chromosome (the one stained only with FITC-dUTP) appears to "lose" it's color, because applying the anti-DNP antibody labeled with FITC, results in much brighter FITC signals. Thus, imaging the FITC channel will permit visualizaton of only the FITC-detected, DNP-dUTP stained chromosomes, which are very bright and require significantly shorter exposure times. The initial FITC-dUTP stained chromosomes, although there, become "invisible", due to their weaker signals. The same principle is applied for other fluors in the CCK algorithm. The final outcome is a procedure allowing differentiation of all 24 different chromosomes using only three fluorescent dyes.
Two variations of the CCK procedure are proposed: the "3+3" strategy uses three fluorescent nucleotides and three haptene-labeled nucleotides (Table 1&2 and Fig. 1&3). The "2+3" or "3+2" strategies use two or three fluorescent-labeled nucleotides and three or two haptene labeled nucleotides, respectively (Table 2 and Fig. 4).
Important note regarding the use of antibodies and the sequence of imaging steps.
After hybridization and the usual posthybridization washes, the slide is subjected to a layer of unlabeled primary antibodies against DIG and DNP, prior to visualization of the signal from the direct, fluor-labeled chromosomes. This protects both haptenes from the destructive effect of short wavelength radiation (particularly through the DAPI filter). BIO-labels are more resistant to photodamage. The slide is mounted with antifade and images of the best metaphases are captured in all three channels. While a CCD camera captures three grayscale images, an RGB digital photographic camera provides three colored images, which are converted into grayscale images using Adobe Photoshop. The three grayscale images are pseudocolored by the computer and a multicolor image is obtained. This is called the "first image" (Fig. 3a, 3d and 4i, 4k), and only chromosomes labeled with fluorescent nucleotides are visible at this time. The coordinates of each metaphase are recorded, a simple task using the verniers of most microscope stages. The antifade-mounting medium is rinsed away and the slide is subjected to appropriate fluorescent-labeled antibodies and avidin (secondary antibodies). Depending on the color combination chosen, the slide may also be subjected to DAPI counterstaining (Fig. 3c). Images of the previously-recorded metaphases are again captured in all three channels, the grayscale images pseudocolored and merged by the computer, yielding the "second image" (Fig. 3b, 3e, 4j, 4l). Some chromosomes fluoresce only in the first or only in the second image and other chromosomes show color changing. Chromosome identification is performed by color comparisons between the two images.
Table 1. Proposed "3+3" CCK chromosome labeling combination.
Table 2. Summary of all "3+3", "2+3" and "3+2" CCK combinations tested.