title: Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry.
abstract: In this paper, we demonstrate the use of synthetic polyamide probes to fluorescently label heterochromatic regions on human chromosomes for discrimination in cytogenetic preparations and by flow cytometry. Polyamides bind to the minor groove of DNA in a sequence-specific manner. Unlike conventional sequence-specific DNA or RNA probes, polyamides can recognize their target sequence without the need to subject chromosomes to harsh denaturing conditions. For this study, we designed and synthesized a polyamide to target the TTCCA-motif repeated in the heterochromatic regions of chromosome 9, Y and 1. We demonstrate that the fluorescently labeled polyamide binds to its target sequence in both conventional cytogenetic preparations of metaphase chromosomes and suspended chromosomes without denaturation. Chromosomes 9 and Y can be discriminated and purified by flow sorting on the basis of polyamide binding and Hoechst 33258 staining. We generate chromosome 9- and Y-specific 'paints' from the sorted fractions. We demonstrate the utility of this technology by characterizing the sequence of an olfactory receptor gene that is duplicated on multiple chromosomes. By separating chromosome 9 from chromosomes 10-12 on the basis of polyamide fluorescence, we determine and differentiate the haplotypes of the highly similar copies of this gene on chromosomes 9 and 11.
authors.affiliation: Department of Molecular Biotechnology, University of Washington, Seattle, WA 98195, USA.
authors.forename: Melanie P
authors.initials: MP
authors.lastname: Gygi
authors.forename: Mark D
authors.initials: MD
authors.lastname: Ferguson
authors.forename: Heather C
authors.initials: HC
authors.lastname: Mefford
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Lund
authors.forename: Christine
authors.initials: C
authors.lastname: O'Day
authors.forename: Peiwen
authors.initials: P
authors.lastname: Zhou
authors.forename: Cynthia
authors.initials: C
authors.lastname: Friedman
authors.forename: Ger
authors.initials: G
authors.lastname: van den Engh
authors.forename: Mark L
authors.initials: ML
authors.lastname: Stolowitz
authors.forename: Barbara J
authors.initials: BJ
authors.lastname: Trask
mesh_terms: D001483:Base Sequence; D006690:Bisbenzimidazole / Q000737:chemistry; D020223:Chromosome Painting; D002877:Chromosomes, Human / Q000235:genetics*; D005434:Flow Cytometry / Q000379:methods; D005453:Fluorescence; D006570:Heterochromatin / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008853:Microscopy / Q000379:methods; D009757:Nylons / Q000737:chemistry*; D009841:Oligonucleotides / Q000235:genetics
chemical_list: D006570:Heterochromatin; D009757:Nylons; D009841:Oligonucleotides; D006690:Bisbenzimidazole