Human Genetics

, Volume 80, Issue 3, pp 224–234 | Cite as

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

  • P. Lichter
  • T. Cremer
  • J. Borden
  • L. Manuelidis
  • D. C. Ward
Original Investigations

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.

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Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • P. Lichter
    • 1
  • T. Cremer
    • 2
  • J. Borden
    • 2
  • L. Manuelidis
    • 2
  • D. C. Ward
    • 1
  1. 1.Department of Human GeneticsYale University School of MedicineNew HavenUSA
  2. 2.Section of NeuropathologyYale University School of MedicineNew HavenUSA

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