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Colour-changing karyotyping: an alternative to M-FISH/SKY

Nature Genetics volume 23, pages 263–264 (1999)Cite this article

The '3+3' combination (Fig. 1a–g) mimics the use of six different 'dyes' and was more robust, primarily because fewer chromosomes require simultaneous labelling with three different dyes/nucleotides. Because FITC, Cy3 and Cy5 are bright, stable and very separable fluors, of the two variations tested, combination 1 yielded more robust fluorescent signals than 2 (Table 1), with signal-to-noise (S/N) ratios from 1.8 to 4.5 for individual chromosomes. DAPI imaging, however, requires a fourth filter. Combination 2 required the use of aminomethyl-coumarin (AMCA), and prevented DAPI counterstaining, as the two dyes have similar spectra. The '2+3' or '3+2' algorithms mimic the use of five 'dyes' ( Fig. 1h–l), and chromosomes were labelled as described3. We compared several labelling combinations (Table 2, 3–6) using a variety of three filter combinations. For general purposes, the most convenient combination is 4, which uses dyes detectable through the three 'standard' filters (DAPI, FITC and rhodamine/Texas Red). 'First image' included FITC, Texas Red and AMCA (avidin), whereas the 'second image' included FITC (DNP), Texas Red (DIG) and DAPI.

We captured images with video/CCD cameras or a digital camera (DP-10, Olympus), replacing the 'classic' film cameras mounted on older-model microscopes. Digital cameras can detect DAPI, FITC and rhodamine signals, which is the minimum requirement for CCK.

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Figure 1: CCK examples.

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Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, Connecticut, USA

    Octavian Henegariu, Patricia Bray-Ward & David C. Ward

  2. Department of Genetics, Hughes Institute, St. Paul, Minnesota, USA

    Nyla A. Heerema

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  1. Octavian Henegariu
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  2. Nyla A. Heerema
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  3. Patricia Bray-Ward
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  4. David C. Ward
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Correspondence to Octavian Henegariu.

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Henegariu, O., Heerema, N., Bray-Ward, P. et al. Colour-changing karyotyping: an alternative to M-FISH/SKY. Nat Genet 23, 263–264 (1999). https://doi.org/10.1038/15437

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