Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy

Thomas Ried, Antonio Baldini, Timothy C. Rand, David C. Ward

Research output: Contribution to journalArticle

375 Citations (Scopus)

Abstract

Combinatorial labeling of probes (i.e., with two or more different reporters) increases the number of target sequences that can be detected simultaneously by fluorescence in situ hybridization. We have used an epifluorescence microscope equipped with a digital imaging camera and computer software for pseudocoloring and merging images to distinguish up to seven different probes using only three fluorochromes. Chromosome-specific centromere repeat clones and chromosome-specific "composite" probe sets were generated by PCR in which different mixtures of modified nucleotides, including fluorescein-conjugated dUTP, were incorporated. Cosmid clones were labeled similarly by nick-translation. The technique has been used to delineate the centromeres of seven different human chromosomes, on both 4′,6-diamidino-2-phenylindole-stained metaphase spreads and interphase nuclei, to map six cosmid clones in a single hybridization experiment and to detect chromosome translocations by chromosome painting. Multiparameter hybridization analysis should facilitate molecular cytogenetics, probe-based pathogen diagnosis, and gene mapping studies.

Original languageEnglish
Pages (from-to)1388-1392
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume89
Issue number4
Publication statusPublished - Feb 15 1992

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Optical Imaging
DNA Probes
In Situ Hybridization
Microscopy
Cosmids
Clone Cells
Centromere
Chromosomes
Chromosome Painting
Molecular Probes
Chromosome Mapping
Interphase
Human Chromosomes
Metaphase
Fluorescein
Fluorescence In Situ Hybridization
Fluorescent Dyes
Cytogenetics
Software
Nucleotides

Keywords

  • CCD camera
  • Centromere repeats
  • Cytogenetics
  • Gene mapping
  • PCR-probe labeling

ASJC Scopus subject areas

  • Genetics
  • General

Cite this

Simultaneous visualization of seven different DNA probes by in situ hybridization using combinatorial fluorescence and digital imaging microscopy. / Ried, Thomas; Baldini, Antonio; Rand, Timothy C.; Ward, David C.

In: Proceedings of the National Academy of Sciences of the United States of America, Vol. 89, No. 4, 15.02.1992, p. 1388-1392.

Research output: Contribution to journalArticle

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