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FISH with complex probes
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General observations
For probes in this category (microdissected probes, whole chromosome paint probes and whole genomic probes) one of the most important parts of the FISH protocol is slide preparation. Cell suspensions used for slide preparation need to be as free of cytoplasmic residua as possible. This requires optimum hypotonic treatment and proper fixation. Metaphases should be spread well enough so that chromosomes do not cross each other, but spreading should not be excessive, so the entire metaphase can fit in a single microscope field (100x magnification). Slide preparation is detailed elsewhere. Chemical aging offers very good results with all complex FISH probes, and dry heat aging of the slide should be avoided. Slides should be prepared fresh and used immediately for hybridization. Slide pretreatment using a protease (usually pepsin) always improved hybridization results. If a better DAPI banding is required (for example in CGH), immediately after the enzymatic pretreatment, the slide can be kept no longer than 15-20 seconds in 1% formaldehyde in isotonic buffer, and then passed through an ethanol series as usual. Too long formaldehyde incubation decreases hybridization. Separate and simultaneous denaturing provide identical results.
Complex probes are conveniently labeled by PCR using degenerate primers. As usual, labeled fragments should be cut to 200-300bp average length before hybridization. Nick translation can also be used for labeling. Complex probes can be mixed with one another, with single-copy probes or with repetitive probes, depending on the purpose. For example, microdissected probes for chromosome 12p, 12q and the 12 centromere probe (Fig. 10k) and microdissected probes for the three bands of 12p were hybridized and detected in triple color FISH (Fig. 10l).
Hybridization time
When using chemical aging, hybridization does not need to proceed longer than overnight (14-16 hours) no matter what probes are used. In our hands, CGH results were identically good after 16 (one day) and 36 hours (2 days) but worse after 70 hours (3 days). M-FISH results were very good after 14-16 hours hybridization. Shorter times were not tested.
Comparative genomic hybridization (CGH)
CGH principle (see figure below). CGH is used to detect differences in DNA copy number between a test sample (for example tumor DNA) and a control sample (normal genomic DNA). The tumor DNA is labeled with a red fluorophore and the normal DNA with a green fluorophore (or vice-versa), and the two samples are mixed together, followed by overnight hybridizen onto normal metaphases. After hybridization and DAPI staining, a computer program is used to calculate the ratio of fluorescence between the red and the green colors along the axis of every chromosome. Whenever the tumor DNA shows DNA amplification in some chromosomal areas, there will be more red-labeled copies of DNA hybridizing for thaose regions, so the hybridization color (and the graph) will shift toward red. Whenever the tumor DNA displays loss of DNA for some chromosomal areas, there will be proportionally more green-labeled fragments hybridizing on those regions, thus the fluorescent color (and graph of ratio) will shift toward green. CGH is particularly important, as it allows to test DNA from archived samples (paraffin embedded material), and does not require cell culture of every test sample.
The quality of slides used for CGH is vital, thus the cell suspension should always be tested. A good cell suspension can be used for over a year, if stored at -20° C in fixative. DAPI banding can be improved by: (1) 10-30 seconds incubation in 1% formaldehyde after pretreatment. (2) gradual slide denaturing, using either the temperature gradient of a thermocycler block or several jars with denaturing solution at various temperatures. (3) DAPI staining in a jar with a DAPI solution, and not by adding DAPI to the antifade solution. DNA labeling can be done by nick translation or PCR (with a degenerate primer). Nick translated probes require, in general, a somewhat larger amount of competitor DNA to compete out repetitive sequences. Fig. 11a shows CGH results when an insufficient amount of competitor DNA was used. The centromeres of almost all chromosomes show bright signals, indicating that repetitive sequences were not competed out. Hybridization of repetitive sequences prevents proper CGH analysis, as they smooth out the differences/ratios between the two colors. In Fig.11b, the DNA was labeled by PCR. Although the DNA fragments were digested by DNase to below 500bp, hybridization was more "grainy". In both Fig. 11a and 11b, posthybridization washing conditions were very stringent (20 minutes at 65° C in 0.2% SSC). Our experiments showed that complex probes do not require high stringency washes. Because repetitive sequences may become a concern, their binding can be avoided by increasing the amount of Cot1 DNA during hybridization. If the hybridization signals along the chromosomes are not smooth, the ratio analysis between the two colors will be less accurate. A good CGH hybridization is depicted in Fig. 11c.
CGH can be performed with DNA probes labeled with fluorescent nucleotides (FITC and TRITC, Fig. 11d-f) or haptene-labeled nucleotides (biotin and digoxigenin, Fig. 11g-i). Both procedures work equally well if labeling is good, DNA fragments are small and slides are properly prepared.
Multicolor karyotyping (M-FISH, SKY and CCK)
Multicolor karyotyping labels and identifies each human chromosome pair by a different fluorescent color, using combinatorial labeling. Two groups (E. Schroeck and M. Speicher), using two different experimental strategies, have pioneered the procedure using five or six different fluors (SKYand M-FISH, respectively). Several M-FISH labeling schemes can be found here. Another group has achieved color karyotyping (Color Changing Karyotyping = CCK) using only three fluorophores, without the need for any specialized equipment or software.
In M-FISH, five fluorophores are used to acquire the desired 24 combinations. Individual chromosome painting libraries and many individual fluorophores were tested, either separately or in groups (Fig. 11j). DNA amount for the various painting probes were adjusted, to bring the signals to comparable intensities. DNA template for all chromosomes labeled with the same dye was mixed together, resulting in five different pools. Five separate labeling reactions (nick translation or PCR) were performed, one on each pool, using the corresponding dye. After labeling, different amounts of each pool were combined and the labeled DNA was precipitated in the presence of Cot1 and resuspended in hybridization buffer. Any five fluorophores can be used for M-FISH, provided that the microscope is equipped with the appropriate set of excitation and emission filters. For example, the original M-FISH procedure used FITC, Cy3, Cy3.5, Cy5 and Cy7. As Cy7 was not available in this laboratory, it was replaced with AMCA (blue side of the spectrum) or Cy5.5 (far-red). Because AMCA and DAPI have similar spectral characteristics, DAPI cannot be used at the same time with AMCA. M-FISH can be performed without DAPI, and various software packages (PSI) can be used to analyze the images even in the absence of DAPI (Fig. 11k). However, if the microscope is equipped with only 3-5 filters and AMCA was used in the analysis, DAPI images can still be captured using the following approach: M-FISH or CCK images of the best metaphases are captured first, and the position of these metaphases recorded using the verniers of the microscope stage. , The coverslip is removed, the slide is rinsed 15 minutes in 4xSSC buffer at room temperature, DAPI staining is performed and images of the same metaphases are captured using the DAPI filter. Depending on the amount of labeled probe used and the cost of individual reagents from a variety of commercial sources, the total cost of one M-FISH hybridization is in the range of $250-300.
However, by custom-preparing fluor-labeled dUTPs (for protocol details click here) and custom labeling the chromosome painting probes , the overall cost per analysis can be reduced to less than $1.
M-FISH examples
1. Detection of balanced or non-balanced translocations.
2. Detection of complex translocation in tumors
