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    SKY or M-FISH and CGH Techniques

Spectral Karyotyping (SKY) and Multiplex Fluorescence In Situ Hybridization (M-FISH)

SKY and M-FISH are molecular cytogenetic techniques that permit the
simultaneous visualization of all human (or mouse) chromosomes in different
colors, considerably facilitating karyotype analysis. Chromosome-specific probe
pools (chromosome painting probes) are generated from flow-sorted chromosomes, and
then amplified and fluorescently labeled by degenerate oligonucleotide-primed
polymerase chain reaction. Both SKY and M-FISH use a combinatorial labeling scheme
with spectrally distinguishable fluorochromes, but employ different methods for detecting
and discriminating the different combinations of fluorescence after in situ hybridization.

In SKY, image acquisition is based on a combination of epifluorescence microscopy,
charge-coupled device (CCD) imaging, and Fourier spectroscopy. This makes possible the
measurement of the entire emission spectrum with a single exposure at all image points.
In M-FISH, separate images are captured for each of the five fluorochromes using
narrow bandpass microscope filters; these images are then combined by dedicated
software. In both techniques, unique pseudo-colors are assigned to the chromosomes
based on their specific fluorochrome signatures.

The applications of SKY and M-FISH for screening genomes for chromosomal aberrations in
human disease and animal models of human cancer are manifold. By making possible the
unambiguous identification of even complex and hidden chromosomal abnormalities, SKY/M-FISH is
particularly useful in :

  • Mapping of chromosomal breakpoints
  • Detection of subtle translocations
  • Identification of marker chromosomes, homogeneously staining
    regions, and double minute chromosomes,
  • Characterization of complex rearrangements.

The notoriously difficult analysis of murine chromosomes has now become greatly simplified,
extending the application of SKY/M-FISH to the visualization of chromosomal aberrations in mouse
models of human cancer.

    Visit the Ried Laboratory WebSite for SKY protocols

Selected SKY/M-FISH References

Comparative Genomic Hybridization (CGH)

Comparative genomic hybridization (CGH) is a fluorescent molecular
cytogenetic technique that identifies DNA gains, losses, and
amplifications, mapping these variations to normal metaphase
chromosomes. It is a powerful tool for screening chromosomal
copy number changes in tumor genomes and has the advantage of
analyzing entire genomes within a single experiment. It is
particularly applicable to the study of tumors which do not
yield sufficient metaphases for cytogenetic analysis and can
be applied to fresh or frozen tissues, cell lines, and archival
formalin-fixed paraffin-embedded samples.

CGH is based on quantitative two-color fluorescence in situ
hybridization. Equal amounts of differentially labeled tumor
genomic DNA and normal reference DNA are mixed together and
hybridized under conditions of Cot-1 DNA suppression to normal
metaphase spreads. The labeled probes are detected with two
different fluorochromes, e.g., FITC for tumor DNA and TRITC
for the normal DNA. The difference in fluorescence intensities
along the chromosomes in the reference metaphase spread are a
reflection of the copy number changes of corresponding sequences
in the tumor DNA.

CGH has the advantage of requiring only genomic tumor DNA, making
it highly useful for cancer cytogenetics, circumventing the need
for high quality tumor metaphase spreads. The ability to study
archival material allows retrospective analysis which can correlate
chromosomal aberrations with the clinical course. Since its
introduction in 1992, CGH has been applied to a broad variety of
tumor types which have previously defied comprehensive cytogenetic
analysis by traditional methods. CGH has, for example:

  • Revealed consistent genetic imbalances and multiple amplification sites in
    carcinomas of the brain, colon, prostate, cervix, and breast. For
    instance, it identified chromosome 7 gain and chromosome 10 loss as
    landmark aberrations in glioblastomas, and specific gains of chromosomes
    1, 8, 17, and 20 and loss of 13q and 17p in breast cancer.
  • Found chromosomal aberrations in human leukemia, lymphoma,
    and solid tumors has identified non-random tumor and tumor-stage
    specific genetic changes.This information can guide positional cloning efforts.
  • Become an important initial screening test for chromosomal
    gains and losses in solid tumor progression, and the results derived
    from these experiments can be applied to the development of more
    specific diagnostics.

            Visit the Ried Laboratory WebSite for CGH protocols

Selected CGH References

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