at interstitial sites (Meyne et al. 1990). Such repeat loci may interfere
with the specificity of the reaction. Preannealing of the probes generally contributes to a higher signal to noise ratio.
5. RNase digestion, which is routinely performed to prevent elevated levels of background fluorescence due to unspecific binding of DNA
probes to RNA molecules in remaining cytoplasm or in nuclei, can
be omitted for metaphase chromosome preparations free ofcytoplasm.
However, it is imperative that preparations of probe DNA are free of
RNA.
6. In the initial Zoo-FISH protocol (Scherthan et al. 1994) probe and target DNAs were denatured simultaneously because this method usually
creates stronger signals. However, separate denaturation is recommended (Rettenberger et al. 1995, Fronicke et al. 1996), since the shearing forces which are created during simultaneous denaturation by the
dextran sulfate polymer of the hybridization solution may cause inferior chromosome morphology.
7. Dehydration of freshly denatured chromosome preparations through
an ethanol series has proven dispensable. It is sufficient to wash down
the coverslip and hybridization solution with a jet of cold deionized
water from a wash bottle and air dry the preparations with an air jet.
8. Formamide containing wash solutions can be replaced effectively with
dilute salt solutions, thereby reducing the handling of formamide to a
few fJ.l.
9. The high amounts of labeled probe required for Zoo-FISH may cause
considerable background levels which impair the use of multi-color
FISH schemes. Generally, it is recommended to perform single or
two color detection since these produce reliable results (Figs. 1,2).
This contrasts with the intra- and intergeneric approach to comparative molecular cytogenetics, where multi-color painting is feasible
(Muller et al. 1998).
10. Comparison of the relative genome fraction delineated with a particular human WCP in a mammalian genome revealed that equivalent
genomic fractions have been maintained during mammalian evolution. It should be noted that values obtained by such measurements
have to be adjusted for the presence of intervening centromeres, heterochromatin blocks or NORs (Scherthan et al. 1994; Chowdhary et al.
1996, Fronicke et al. 1996,1997).
25 Zoo-FISH
319
with the specificity of the reaction. Preannealing of the probes generally contributes to a higher signal to noise ratio.
5. RNase digestion, which is routinely performed to prevent elevated levels of background fluorescence due to unspecific binding of DNA
probes to RNA molecules in remaining cytoplasm or in nuclei, can
be omitted for metaphase chromosome preparations free ofcytoplasm.
However, it is imperative that preparations of probe DNA are free of
RNA.
6. In the initial Zoo-FISH protocol (Scherthan et al. 1994) probe and target DNAs were denatured simultaneously because this method usually
creates stronger signals. However, separate denaturation is recommended (Rettenberger et al. 1995, Fronicke et al. 1996), since the shearing forces which are created during simultaneous denaturation by the
dextran sulfate polymer of the hybridization solution may cause inferior chromosome morphology.
7. Dehydration of freshly denatured chromosome preparations through
an ethanol series has proven dispensable. It is sufficient to wash down
the coverslip and hybridization solution with a jet of cold deionized
water from a wash bottle and air dry the preparations with an air jet.
8. Formamide containing wash solutions can be replaced effectively with
dilute salt solutions, thereby reducing the handling of formamide to a
few fJ.l.
9. The high amounts of labeled probe required for Zoo-FISH may cause
considerable background levels which impair the use of multi-color
FISH schemes. Generally, it is recommended to perform single or
two color detection since these produce reliable results (Figs. 1,2).
This contrasts with the intra- and intergeneric approach to comparative molecular cytogenetics, where multi-color painting is feasible
(Muller et al. 1998).
10. Comparison of the relative genome fraction delineated with a particular human WCP in a mammalian genome revealed that equivalent
genomic fractions have been maintained during mammalian evolution. It should be noted that values obtained by such measurements
have to be adjusted for the presence of intervening centromeres, heterochromatin blocks or NORs (Scherthan et al. 1994; Chowdhary et al.
1996, Fronicke et al. 1996,1997).
25 Zoo-FISH
319