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Nucleic Acids Research, 2002, Vol. 30, No. 13 2790-2799
© 2002 Oxford University Press

Use of fluorescent sequence-specific polyamides to discriminate human chromosomes by microscopy and flow cytometry

Melanie P. Gygi1, Mark D. Ferguson3, Heather C. Mefford2,4, Kevin P. Lund3, Christine O’Day3, Peiwen Zhou3, Cynthia Friedman4, Ger van den Engh5, Mark L. Stolowitz3 and Barbara J. Trask1,2,4,*

1 Department of Molecular Biotechnology and 2 Department of Genetics, University of Washington, Seattle, WA 98195, USA, 3 Prolinx Inc., Bothell, WA 98021, USA, 4 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA and 5 Institute for Systems Biology, Seattle, WA 98103, USA

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.

* To whom correspondence should be addressed at: Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, PO Box 19024, Mailstop C3-168, Seattle, WA 98109-1024, USA. Tel: +1 206 667 1470; Fax: +1 206 667 4023; Email: btrask{at}fhcrc.org Present addresses:Melanie P. Gygi, Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USAChristine O’Day, CEPTYR, Inc., Bothell, WA 98021, USAPeiwen Zhou, Exelixis, Inc., South San Francisco, CA 94083, USAMark L. Stolowitz, LumiCyte, Inc., Fremont, CA 94538, USA


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