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Multiplex-fluorescence in situ hybridization for chromosome karyotyping

Nature Protocols volume 1, pages 1172–1184 (2006)Cite this article

Abstract

Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes — the 22 autosomes and X and Y sex chromosomes — with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes ∼14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use.

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Figure 1: Possible labeling schemes for human (a,b) and mouse (c,d) M-FISH probe mixes.
Figure 2: Exemplary DOP-PCR amplification product on a 1% (wt/vol) agarose gel.
Figure 3: DOP-PCR labeling amplification products on a 1% (wt/vol) agarose gel before (a) and after (b) DNAse I digestion.
Figure 4: Human male metaphase spread (a) and karyotype (b) after hybridization of a seven-fluorochrome M-FISH mix.

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Authors and Affiliations

  1. Institute of Medical Biology and Human Genetics, Medical University of Graz, Harrachgasse 21/8, Graz, A-8010, Austria

    Jochen B Geigl, Sabine Uhrig & Michael R Speicher

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  1. Jochen B Geigl
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  2. Sabine Uhrig
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  3. Michael R Speicher
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Correspondence to Michael R Speicher.

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Geigl, J., Uhrig, S. & Speicher, M. Multiplex-fluorescence in situ hybridization for chromosome karyotyping. Nat Protoc 1, 1172–1184 (2006). https://doi.org/10.1038/nprot.2006.160

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