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MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
Subprotocol 9
Multicolor Fluorescence in situ Hybridization
FISH allows the simultaneaus detection of multiple target sequences. The
targets are marked with differentially labeled DNA probes which are visualized via different fluorochromes. For multicolor hybridization experiments, only two steps of the standard protocols must be modified.
Procedure
Multicolor 1. Instead of only one probe, several probes coupled with different reporter
fluorescence
molecules are combined prior to DNA precipitation (Subprotocol 4.).
2. The appropriate reporter binding molecules must be added to the detection buffer (Subprotocol 6). Each reporter binding molecule must be
conjugated with a fluorochrome that can be spectrally distinguished
from other fluorochromes used in the same experiment.
The number of targets that can be simultaneously visualized by multicolor
FISH can be increased when the probes are modified by different reporter
groups and detected by spectrally distinct fluorochromes. Such combinatoriallabelling was demonstrated by several groups (Ried et al. 1992; Wiegantet al. 1991; Dauwerse et al. 1992; Nederlof et al. 1992). In addition, the
combined use of two fluorochromes at different ratios further increases the
number of simultaneously detected sequences (Ried et al. 1992; N ederlof et
al. 1992). Using the combinatorial labeling approach with five different
fluorescent dyes, the simultaneaus visualization of each human chromosome in a specific color has become possible (so called "MultiplexFISH": Speicher et al., 1996; or "Spectral Karyotyping" Schröck et al.,
1996). For the first time, these techniques provide a general screening
test for chromosomal aberrations in a single experiment (Lichter 1997).
Comments
Microscopy
Signals from painted whole chromosomes, chromosomal subregions and
single probes as small as 5 -10 kb arevisible with a conventional fluorescence microscope. Special attention should be paid to the selection of ap-
MARTIN BENTZ, STEFAN JOOS AND PETER LICHTER
Subprotocol 9
Multicolor Fluorescence in situ Hybridization
FISH allows the simultaneaus detection of multiple target sequences. The
targets are marked with differentially labeled DNA probes which are visualized via different fluorochromes. For multicolor hybridization experiments, only two steps of the standard protocols must be modified.
Procedure
Multicolor 1. Instead of only one probe, several probes coupled with different reporter
fluorescence
molecules are combined prior to DNA precipitation (Subprotocol 4.).
2. The appropriate reporter binding molecules must be added to the detection buffer (Subprotocol 6). Each reporter binding molecule must be
conjugated with a fluorochrome that can be spectrally distinguished
from other fluorochromes used in the same experiment.
The number of targets that can be simultaneously visualized by multicolor
FISH can be increased when the probes are modified by different reporter
groups and detected by spectrally distinct fluorochromes. Such combinatoriallabelling was demonstrated by several groups (Ried et al. 1992; Wiegantet al. 1991; Dauwerse et al. 1992; Nederlof et al. 1992). In addition, the
combined use of two fluorochromes at different ratios further increases the
number of simultaneously detected sequences (Ried et al. 1992; N ederlof et
al. 1992). Using the combinatorial labeling approach with five different
fluorescent dyes, the simultaneaus visualization of each human chromosome in a specific color has become possible (so called "MultiplexFISH": Speicher et al., 1996; or "Spectral Karyotyping" Schröck et al.,
1996). For the first time, these techniques provide a general screening
test for chromosomal aberrations in a single experiment (Lichter 1997).
Comments
Microscopy
Signals from painted whole chromosomes, chromosomal subregions and
single probes as small as 5 -10 kb arevisible with a conventional fluorescence microscope. Special attention should be paid to the selection of ap-