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 enzyme-labeled 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 laser-scanning 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.
Lichter
P
P
Department of Human Genetics, Yale University School of Medicine, New Haven, CT 06510.
Cremer
T
T
Borden
J
J
Manuelidis
L
L
Ward
D C
DC
D002877:Chromosomes, Human / Q000648:ultrastructure*; D015342:DNA Probes; D004274:DNA, Recombinant; D006801:Humans; D007399:Interphase; D007621:Karyotyping; D008677:Metaphase; D009693:Nucleic Acid Hybridization
D015342:DNA Probes; D004274:DNA, Recombinant