Abstract
The public human genome sequencing project utilized a hierarchical approach. A large number of BAC/PAC clones, with an insert size approximate from 50 kb to 300 kb, were identified and finely mapped with respect to the Sequence Tagged Site (STS) physical map and with respect to each other. A “golden path” of BACs, covering the entire human genome, was then selected and each clone was fully sequenced. The large number of remaining BACs was not fully sequenced, but the availability of the end sequence (~800–1000 bp) at each end allowed them to be very precisely mapped on the human genome.
The search for copy number variations of the human genome used several strategies. One of these approaches took advantage of the fact that fosmid clones, contrary to BAC/PAC clones, have a fixed insert size (~40 kb) (Kidd et al., Nature 453: 56–64, 2008). In this context, the ends of ~7 million fosmid clones were sequenced, and therefore it was possible to precisely map these clones on the human genome.
In summary, a large number of genomic clones (GC) are available for FISH experiments. They usually yield bright FISH signals and are extremely precious for molecular cytogenetics, and in particular cancer cytogenetics. The already-labeled probes available commercially are usually based on a combination of such GCs. The present chapter summarizes the protocols for extracting, labeling, and hybridization onto slides of DNA obtained from GC.
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References
Kidd JM, Cooper GM, Donahue WF et al (2008) Mapping and sequencing of structural variation from eight human genomes. Nature 453:56–64
Henegariu O, Bray-Ward P, Ward DC (2000) Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling. Nat Biotechnol 18:345–348
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Tolomeo, D., Stanyon, R.R., Rocchi, M. (2017). Fluorescence In Situ Hybridization Probe Preparation. In: Wan, T. (eds) Cancer Cytogenetics. Methods in Molecular Biology, vol 1541. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6703-2_9
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DOI: https://doi.org/10.1007/978-1-4939-6703-2_9
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