title: Robust 3D DNA FISH using directly labeled probes.
abstract: 3D DNA FISH has become a major tool for analyzing three-dimensional organization of the nucleus, and several variations of the technique have been published. In this article we describe a protocol which has been optimized for robustness, reproducibility, and ease of use. Brightly fluorescent directly labeled probes are generated by nick-translation with amino-allyldUTP followed by chemical coupling of the dye. 3D DNA FISH is performed using a freeze-thaw step for cell permeabilization and a heating step for simultaneous denaturation of probe and nuclear DNA. The protocol is applicable to a range of cell types and a variety of probes (BACs, plasmids, fosmids, or Whole Chromosome Paints) and allows for high-throughput automated imaging. With this method we routinely investigate nuclear localization of up to three chromosomal regions.
authors: Nuclear Dynamics Programme, The Babraham Institute. daniel.bolland@babraham.ac.uk
authors: Daniel J
authors: DJ
authors: Bolland
authors: Michelle R
authors: MR
authors: King
authors: Wolf
authors: W
authors: Reik
authors: Anne E
authors: AE
authors: Corcoran
authors: Christel
authors: C
authors: Krueger
mesh_terms: D000498:Allyl Compounds / Q000737:chemistry; D000818:Animals; D004247:DNA / Q000737:chemistry*; D015342:DNA Probes / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000737:chemistry; D021621:Imaging, Three-Dimensional / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D051379:Mice; D014544:Uridine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry
chemical_list: C524080:5-(aminoallyl)-2'-deoxyuridine 5'-triphosphate; D000498:Allyl Compounds; D015342:DNA Probes; D005456:Fluorescent Dyes; D004247:DNA; D014544:Uridine Triphosphate
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