title: Comprehensive FISH probe design tool applied to imaging human immunoglobulin class switch recombination.
abstract: We present a web engine boosted fluorescence in-situ hybridization (webFISH) algorithm using a genome-wide sequence similarity search to design target-specific single-copy and repetitive DNA FISH probes. The webFISH algorithm featuring a user-friendly interface (http://www.webfish2.org/) maximizes the coverage of the examined sequences with FISH probes by considering locally repetitive sequences absent from the remainder of the genome. The highly repetitive human immunoglobulin heavy chain sequence was analyzed using webFISH to design three sets of FISH probes. These allowed direct simultaneous detection of class switch recombination in both immunoglobulin-heavy chain alleles in single cells from a population of cultured primary B cells. It directly demonstrated asynchrony of the class switch recombination in the two alleles in structurally preserved nuclei while permitting parallel readout of protein expression by immunofluorescence staining. This novel technique offers the possibility of gaining unprecedented insight into the molecular mechanisms involved in class switch recombination.
authors.affiliation: Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom. jakub.nedbal@kcl.ac.uk
authors.forename: Jakub
authors.initials: J
authors.lastname: Nedbal
authors.forename: Philip S
authors.initials: PS
authors.lastname: Hobson
authors.forename: David J
authors.initials: DJ
authors.lastname: Fear
authors.forename: Rainer
authors.initials: R
authors.lastname: Heintzmann
authors.forename: Hannah J
authors.initials: HJ
authors.lastname: Gould
mesh_terms: D019295:Computational Biology / Q000379:methods; D015342:DNA Probes; D006801:Humans; D007091:Image Processing, Computer-Assisted; D017578:Immunoglobulin Class Switching; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D017404:In Situ Hybridization, Fluorescence; D020407:Internet; D011995:Recombination, Genetic; D012984:Software; D014584:User-Computer Interface
chemical_list: D015342:DNA Probes; D007143:Immunoglobulin Heavy Chains