318
HARRySCHERTHAN
12. Apply 18 III antifade solution containing DAPI as DNA specific counterstain and mount a 22x60 mm coverslip.
13. Cover slides with a tissue and apply gentle pressure to remove excess
liquid and trapped air bubbles. Inspect under fluorescence microscope
equipped with appropriate filter sets for the visualization of green, red
and blue fluorescence. Images may be recorded with conventional microphotography or digital image acquisition systems.
Troubleshooting
1. Since the quality of the chromosome preparations is crucial for success
of a Zoo-FISH experiment, fixation of the cell pellet at least five times
with freshly prepared ice cold fixative (methanol/acetic acid, 3/1)
should be performed. This should produce metaphase spreads free
of cytoplasm with a crisp appearance upon DAPI staining (Fig. 1). Thymidine synchronization and BrdU incorporation (Drouin et al. 1990,
Hayes et al. 1991) may be applied to obtain long chromosomes which
facilitate FISH mapping due to enhanced DAPI contrast (Carter 1994,
Fronicke and Scherthan 1997).
2. A variable performance of human WCPs in Zoo-FISH experiments has
been noted (Fronicke 1997). Therefore, probe concentrations in the
range of 200-330 ng/Ill have to be tested. In contrary to PBS libraries,
which contain vector sequences, PCR generated probes contain negligible amounts of vector/adaptor sequences which allows them to
be used at lower concentrations (150-200 ng/Ill). In some cases, e.g.
HSA 11, probes from other sources are the key to a successful experiment (Goureau et al. 1996, Fronicke et al. 1997, Dixkens et al. 1998). It
should be noted that WCPs of the human Y chromosome generally fail
to delineate corresponding segments of conserved synteny in ZooFISH experiments (Chowdhary et al. 1998).
3. When pre-labeled commercial WCP probes are used in Zoo-FISH, hybridization times have to be extended to > 7 days (Rettenberger et al.
1995, Goureau et al. 1996).
4. Suppression DNA (usually the Cot! fraction of the genome from which
the paint probe has been generated) is added to the Zoo-FISH hybridization solution, since some repetitive DNA sequences may be shared
among genomes (e.g. telomere repeat sequence motifs have been
found in satellite DNA of Rodents and Cetaceans, or may be located
HARRySCHERTHAN
12. Apply 18 III antifade solution containing DAPI as DNA specific counterstain and mount a 22x60 mm coverslip.
13. Cover slides with a tissue and apply gentle pressure to remove excess
liquid and trapped air bubbles. Inspect under fluorescence microscope
equipped with appropriate filter sets for the visualization of green, red
and blue fluorescence. Images may be recorded with conventional microphotography or digital image acquisition systems.
Troubleshooting
1. Since the quality of the chromosome preparations is crucial for success
of a Zoo-FISH experiment, fixation of the cell pellet at least five times
with freshly prepared ice cold fixative (methanol/acetic acid, 3/1)
should be performed. This should produce metaphase spreads free
of cytoplasm with a crisp appearance upon DAPI staining (Fig. 1). Thymidine synchronization and BrdU incorporation (Drouin et al. 1990,
Hayes et al. 1991) may be applied to obtain long chromosomes which
facilitate FISH mapping due to enhanced DAPI contrast (Carter 1994,
Fronicke and Scherthan 1997).
2. A variable performance of human WCPs in Zoo-FISH experiments has
been noted (Fronicke 1997). Therefore, probe concentrations in the
range of 200-330 ng/Ill have to be tested. In contrary to PBS libraries,
which contain vector sequences, PCR generated probes contain negligible amounts of vector/adaptor sequences which allows them to
be used at lower concentrations (150-200 ng/Ill). In some cases, e.g.
HSA 11, probes from other sources are the key to a successful experiment (Goureau et al. 1996, Fronicke et al. 1997, Dixkens et al. 1998). It
should be noted that WCPs of the human Y chromosome generally fail
to delineate corresponding segments of conserved synteny in ZooFISH experiments (Chowdhary et al. 1998).
3. When pre-labeled commercial WCP probes are used in Zoo-FISH, hybridization times have to be extended to > 7 days (Rettenberger et al.
1995, Goureau et al. 1996).
4. Suppression DNA (usually the Cot! fraction of the genome from which
the paint probe has been generated) is added to the Zoo-FISH hybridization solution, since some repetitive DNA sequences may be shared
among genomes (e.g. telomere repeat sequence motifs have been
found in satellite DNA of Rodents and Cetaceans, or may be located