Multi‐Color FISH Techniques
Traditional FISH analysis has employed, at most, two colors of detection, a red‐fluorescing fluorochrome and a green‐fluorescing fluorochrome. The improvements in fluorescent imaging and development of heartier fluorochromes/dyes have enabled investigators to use several different DNA probes in one experiment. This may involve all or combinations of locus‐specific probes and chromosome paints. The value of such experiments lies in the investigator obtaining far more information from one specific cell at one time, rather then carrying out separate experiments on multiple specimens prepared from the same sample, then extrapolating results. The generic term for multi‐color FISH assays is M‐FISH, however, the technologies behind the manner in which the fluorochrome information is generated has spawned two different M‐FISH systems: spectral karyotyping (SKY) and M‐FISH. For both assays, the experimental procedures are identical: commercially available probes for all 24 (human) chromosomes are differentially labeled according to a labeling scheme and hybridized to metaphase spreads for 24 to 48 hr, followed by post‐hybridization washes and, if required, antibody detection. The difference lies in the imaging: spectral karyotyping identifies the differentiation of the chromosomes based on their spectral properties, whereas M‐FISH identifies the differentiation of the chromosomes based on that fluorochrome's presence or absence when visualized with specific filters. The resulting analysis for both methods is the same, revealing hidden translocations and insertions as well as the chromosomal components of marker chromosomes.
Keywords: FISH; SKY; M‐FISH; hybridization; whole‐chromosome paints; locus‐specific probe
Table of Contents
- Basic Protocol 1: Labeling Whole‐Chromosome DNA Probes for Multi‐Color FISH Assays
- Basic Protocol 2: In Situ Hybridization for Spectral Karyotyping (SKY)
- Alternate Protocol 1: In Situ Hybridization for M‐FISH Karyotyping
- Alternate Protocol 2: Pretreatment of Previously G‐Banded Slides for SKY OR M‐FISH
- Alternate Protocol 3: FISH Analysis Using Locus‐Specific or Chromosome Painting Probes Following SKY/M‐FISH Hybridization
- Reagents and Solutions
- Literature Cited
Basic Protocol 1: Labeling Whole‐Chromosome DNA Probes for Multi‐Color FISH Assays
Basic Protocol 2: In Situ Hybridization for Spectral Karyotyping (SKY)
Alternate Protocol 1: In Situ Hybridization for M‐FISH Karyotyping
Alternate Protocol 2: Pretreatment of Previously G‐Banded Slides for SKY OR M‐FISH
Alternate Protocol 3: FISH Analysis Using Locus‐Specific or Chromosome Painting Probes Following SKY/M‐FISH Hybridization
Figure 22.5.5 The results of sequential G‐banding (A) and SKY (B) analysis of a rhabdomyoscaroma cell line.
Figure 22.5.6 The results of sequential G‐banding (A), SKY (B), and locus‐specific FISH (C) for the BCR‐ABL translocation in a CML sample.
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