Flourescence In-Situ Hybridisation [FISH]
FISH is an important molecular cytogenetic tool used for the identification of genetic disorders and the detection of chromosomal [DNA] genetic abnormalities. The in-situ hybridisation technique involves making the nucleic acid in the target chromosomes or nuclei single stranded and incubating it with a short [e.g. 3kbp] single stranded complimentary probe.
With flourescence in-situ hybridisation [FISH], the probe labels may be fluorochromes or other molecules that are subsequently detected with flourescent affinity reagents. After hybridisation, the numbers, intensity and spatial distribution of the fluorecent signals are analysed. This gives an understanding of how the target nucleic acid sequences are distributed within the cells and chromosomes.
Metaphase and Interphase FISH
Metaphase FISH is used to map the location of a DNA sequence on a chromosome, using fluorescence microscopy. Metaphase FISH identifies the position of DNA sequences ranging in length from IMAGE clones to YACs. Chimerism and sequence homology can also be established. The location of integrated sequences can be determined via metaphase FISH using probes made from the transfected products in combination with chromosome banding or painting. Two, three and four colour FISH is used to determine clone order. Similarly, the DNA probe sequence can also be visualised within the cell nucleus using interphase FISH.
FISH to DNA fibres with BAC and PAC allows rapid visualisation of the extent of overlaps and gaps in contigs. Small clones can be hybridised against larger YAC clones to confirm clone order over longer distances. Fibre FISH with transfected clones allows the quantitation of integrated sequences and can be used to demonstrate the final integrity of the construct.
Choice and resolution of FISH techniques
- Metaphase FISH resolves probes over approximately 2Mbp apart and is suitable for chromosome and band assignment
- Interphase FISH resolves probes 100Kbp-2Mbp apart and is suitable for clone ordering and orientation
- Fibre FISH resolves probes 10Kbp-500Kbp apart and is suitable for clone ordering and to assess overlaps and gaps in contig maps
M-FISH and CGH
Multiplex Fluorescence In Situ Hybridization (M-FISH) and Comparative Genomic Hybridization (CGH) are complementary fluorescent molecular cytogenetic techniques. M-FISH acquires images of each human or mouse chromosome in a different color, allowing semi-automatic karytyping and the identification of chromosomal aberrations.
The complimentary technique of CGH visualises the hybridization of differentially labeled reference DNA sequences to generate a high resolution map of DNA copy number changes in the genome. The WTCHG can further investigate copy number variation using the Affymetrix GeneChip Cytogenetics Array system.
In addition there are routine classical cytogenetics techniques like Giesma DNA staining for chromosome banding image acquistion and semi-automatic human chromosome karyoyping.
All images: The Molecular Cytogenetics Group and the Microscopy Core, WTCHG.
For more details on molecular cytogenetics theory and techniques check out our website links.