Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries

A method of in situ hybridization for visualizing individual human
chromosomes from pter to qter, both in metaphase spreads and
interphase nuclei, is reported. DNA inserts from a single
chromosomal library are labeled with biotin and partially
preannealed with a titrated amount of total human genomic DNA prior
to hybridization with cellular or chromosomal preparations. The
cross-hybridization of repetitive sequences to nontargeted
chromosomes can be markedly suppressed under appropriate
preannealing conditions. The remaining single-stranded DNA is
hybridized to specimens of interest and detected with fluorescent
or enzymelabeled avidin conjugates following post-hybridization
washes. DNA inserts from recombinant libraries for chromosomes 1,
4, 7, 8, 13, 14, 18, 20, 21, 22, and X were assessed for their
ability to decorate specifically their cognate chromosome; most
libraries proved to be highly specific. Quantitative densitometric
analyses indicated that the ratio of specific to nonspecific
hybridization signal under optimal preannealing conditions was at
least 8:1. Interphase nuclei showed a cohesive territorial
organization of chromosomal domains, and laserscanning confocal
fluorescence microscopy was used to aid the 3-D visualization of
these domains. This method should be useful for both karyotypic
studies and for the analysis of chromosome topography in interphase
cells.