Fluorescence in Situ Hybridization, Fluorochromes, Fluorescent Molecules, Yeast Artificial Chromosome,Cosmid Subclones

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	Fluorescence in Situ Hybridization The construction of molecular or physical maps requires continued isolation of new DNA fragments and their evaluation as markers. The first step in this direction is in situ hybridization. In situ hybridization involves hybridization of suitably labelled probes (DNA or RNA) with specially prepared chromosomes in squash preparations fixed to microscopic slides.
Initially, radiolabelled probes were used, but currently probes tagged with fluorescent molecules (fluorescence-labelled) are being used. The chromosomes held on slides are suitably pretreated to expose and denature their DNAs without affecting their structural integrity. The chromosomes are loaded with the labelled probe, incubated under annealing conditions, washed to remove the unhybridized probe, and the location of hybridized probe in the chromosomes is determined by using autoradiography (radio-labelled probes) (Appendix 2.VII) or fluorescence microscopy (fluorescence-labelled probes).
Fluorescence in situ hybridization (FISH) uses fluorescence labelled probes of 1 kb or greater size, usually 40 kb or larger, for in situ hybridization (see, box). Different fluorochromes (fluorescent molecules), each giving a different colour, can be tagged to different probes all of which can be used at the same time in the same preparation.
When several such probes specific to a single chromosome are used in a preparation their location in the chromosome with respect to each other can be determined. In case of metaphase preparations, the probes have to be located at least 1 Mb (megabase pairs, 106 bp) apart for distinction. Further, two already mapped probes are used in conjunction with a third probe whose order is to be determined. FISH has played a great role in the correct ordering of Y AC (yeast artificial chromosome) contigs along the chromosome, and in ordering of cosmid subclones within a single YAC clone.