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

Abstract

Summary.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.

Cytogenetic abnormalities in 1162 couples with recurrent miscarriages in Southern region of India: report and review

“…FISH (Fluorescence in situ hybridization) was performed using commercially available (WCP) whole chromosomepainting probes for chromosomes 3, 14, 18 and 22 (Kreatech). Chromosome denaturation, hybridization and signal detection were done according to the published protocols [6,7].…”

Interplay between ATM and ATR in the regulation of common fragile site stability

“…Probes were labeled as previously described (Schwartz et al, 2005). FISH on metaphase chromosomes was performed as previously described (Lichter et al, 1988).…”

Identification of iso(18p) marker chromosome by fluorescencein situ hybridization with single-copy DNA probe

“…FISH was performed according to the methods described elsewhere (Lichter et al, 1988;Takahashi et al, 1991). Cosmid DNAs were labeled by nick translation using the enzyme kit (Boehringer Mannheim, GmbH) with biotin-ll-dUTP (Boehringer Mannheim) and hybridized to chromosome DNA under conditions that suppress signals from repetitive sequences (Lichter et aL, 1988).…”