18
EIGIL KJELDSEN and STEEN K0LVRAA
phatase-system and the peroxidase system where the enzymes are coupled
to nucleotides, or more commonly, steptavidin or antibodies. For detection, a substrate is then added which by the enzymes action forms a precipitate at the site of annealing. This, in turn, can be detected by light microscopy. The alkaline phosphatase system is considered to be more sensitive than the peroxidase system, which on the other hand tends to give
greater resolution.
When isotopic labels are utilized, a dip-slide emulsion technique is
used, followed by light microscopy as in the enzyme-based systems.
Refinements of the standard FISH technique
Reverse cytogenetics
The possibilities of isolating chromosomes by flow-sorting (Carter et al.
1992) or by microdissection (Thangavelu et al. 1994) have been used to
isolate aberrant, but unidentified chromosomes. After isolation of the
aberrant chromosome it is amplified, labeled and used as a probe on metaphases from a normal individual. This technique is called reverse cytogenetics or reverse painting and is used to determine the composition of
the aberrant chromosome (Carter et al. 1992).
Multicolor FISH technology
FISH has the capacity to simultaneously visualize different targets in multiple, distinct colors. The simplest form of multicolor FISH is to mix differently labeled probes in a single hybridization solution. However, due to
spectral overlap of the fluorochromes, the number of probes identifiable
on the basis of a unique fluorescence color is limited to three for the visible
part of the spectrum, and to five if far-red and infra-red fluorescing dyes
are included. When a more sophisticated approach such as combinatorial
labeling is used, the different probes are labeled with combinations ofhaptens or fluorochromes and the number of identifiable targets can be increased (Nederlof et al. 1990, Ried et al., 1992, Wiegant et al. 1993). In
principle, the number of targets identifiable on the basis of a unique, binary combination is 2n-l, where n is the number of different labels. Thus, if
five fluorochromes are included, 31 different combinations can be obtained. Another approach is ratio-labeling where varying ratios of different fluorochromes are used for each probe so that it is the fluorescence
EIGIL KJELDSEN and STEEN K0LVRAA
phatase-system and the peroxidase system where the enzymes are coupled
to nucleotides, or more commonly, steptavidin or antibodies. For detection, a substrate is then added which by the enzymes action forms a precipitate at the site of annealing. This, in turn, can be detected by light microscopy. The alkaline phosphatase system is considered to be more sensitive than the peroxidase system, which on the other hand tends to give
greater resolution.
When isotopic labels are utilized, a dip-slide emulsion technique is
used, followed by light microscopy as in the enzyme-based systems.
Refinements of the standard FISH technique
Reverse cytogenetics
The possibilities of isolating chromosomes by flow-sorting (Carter et al.
1992) or by microdissection (Thangavelu et al. 1994) have been used to
isolate aberrant, but unidentified chromosomes. After isolation of the
aberrant chromosome it is amplified, labeled and used as a probe on metaphases from a normal individual. This technique is called reverse cytogenetics or reverse painting and is used to determine the composition of
the aberrant chromosome (Carter et al. 1992).
Multicolor FISH technology
FISH has the capacity to simultaneously visualize different targets in multiple, distinct colors. The simplest form of multicolor FISH is to mix differently labeled probes in a single hybridization solution. However, due to
spectral overlap of the fluorochromes, the number of probes identifiable
on the basis of a unique fluorescence color is limited to three for the visible
part of the spectrum, and to five if far-red and infra-red fluorescing dyes
are included. When a more sophisticated approach such as combinatorial
labeling is used, the different probes are labeled with combinations ofhaptens or fluorochromes and the number of identifiable targets can be increased (Nederlof et al. 1990, Ried et al., 1992, Wiegant et al. 1993). In
principle, the number of targets identifiable on the basis of a unique, binary combination is 2n-l, where n is the number of different labels. Thus, if
five fluorochromes are included, 31 different combinations can be obtained. Another approach is ratio-labeling where varying ratios of different fluorochromes are used for each probe so that it is the fluorescence