Chapter 31
PROTOCOL
Multicolor FISH Techniques for the Detection
of Inter- and Intrachromosomal Rearrangements
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Introduction
Fluorescence in situ hybridization (FISH) technology has evolved constantly during the past decade. Nowadays, a large variety of probes detected with novel fluorescent dyes, allows the simultaneous detection
of several different nucleic acid targets. A simultaneous hybridization
of different probes has led to "multicolor FISH" experiments in which
more than two fluorescent dyes are used for probe detection. Probes
of greatly differing complexity can be combined for multicolor FISH procedures, including whole or partial chromosome painting probes, locusspecific probes (YAC, BAC, PI, cosmids) and repetitive sequences. Since
only a limited number of distinct fluorescent dyes can be resolved with the
standard microscope setup, combinatorial labeling schemes have been developed. In most circumstances, this wide variety of mixed colors can only
be distinguished by utilisation of digital image acquisition and analysis
systems. Multicolor FISH procedures are also demonstrated elsewhere
in this manual describing for instance the simultaneous staining of
each chromosome homologue in the human or murine chromosome complement in a different color (Chapter 22). In this chapter, protocols are
presented describing the application of multicolor FISH in radiation
~ Horst Zitzelsberger, GSF-Forschungszentrum, lnstitut fUr Strahlenbiologie,
Ingolstadter Landstr. 1, OberscWeiGheim, 85764, Germany (phone +49-89-31873421;
fax +49-89-31872873; e-mail Zitzelsberger@gsf.de), Ludwig-Maximilians-Universitat,
Institute of Radiation Biology, Miinchen, 80336, Germany
Benjamin O'Brien, University of California, Department of Subcellular Structures, Life
Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, 94720,
USA
Heinz-Ulrich Weier, University of California, Department of Subcellular Structures,
Life Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA,
94720, USA
PROTOCOL
Multicolor FISH Techniques for the Detection
of Inter- and Intrachromosomal Rearrangements
HORST ZITZELSBERGER, BENJAMIN O'BRIEN, and HEINZ-ULRICH WEIER
Introduction
Fluorescence in situ hybridization (FISH) technology has evolved constantly during the past decade. Nowadays, a large variety of probes detected with novel fluorescent dyes, allows the simultaneous detection
of several different nucleic acid targets. A simultaneous hybridization
of different probes has led to "multicolor FISH" experiments in which
more than two fluorescent dyes are used for probe detection. Probes
of greatly differing complexity can be combined for multicolor FISH procedures, including whole or partial chromosome painting probes, locusspecific probes (YAC, BAC, PI, cosmids) and repetitive sequences. Since
only a limited number of distinct fluorescent dyes can be resolved with the
standard microscope setup, combinatorial labeling schemes have been developed. In most circumstances, this wide variety of mixed colors can only
be distinguished by utilisation of digital image acquisition and analysis
systems. Multicolor FISH procedures are also demonstrated elsewhere
in this manual describing for instance the simultaneous staining of
each chromosome homologue in the human or murine chromosome complement in a different color (Chapter 22). In this chapter, protocols are
presented describing the application of multicolor FISH in radiation
~ Horst Zitzelsberger, GSF-Forschungszentrum, lnstitut fUr Strahlenbiologie,
Ingolstadter Landstr. 1, OberscWeiGheim, 85764, Germany (phone +49-89-31873421;
fax +49-89-31872873; e-mail Zitzelsberger@gsf.de), Ludwig-Maximilians-Universitat,
Institute of Radiation Biology, Miinchen, 80336, Germany
Benjamin O'Brien, University of California, Department of Subcellular Structures, Life
Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA, 94720,
USA
Heinz-Ulrich Weier, University of California, Department of Subcellular Structures,
Life Sciences Division, E. O. Lawrence Berkeley National Laboratory, Berkeley, CA,
94720, USA