Human Genetics

, Volume 80, Issue 3, pp 224–234

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.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

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
  3. 3.Institut für Humangenetik und Anthropologie der UniversitätHeidelberg 1Federal Republic of Germany

Personalised recommendations