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Combinatorial Oligo FISH: Directed Labeling of Specific Genome Domains in Differentially Fixed Cell Material and Live Cells

  • Eberhard Schmitt
  • Jutta Schwarz-Finsterle
  • Stefan Stein
  • Carmen Boxler
  • Patrick Müller
  • Andriy Mokhir
  • Roland Krämer
  • Christoph Cremer
  • Michael HausmannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 659)

Abstract

With the improvement and completeness of genome databases, it has become possible to develop a novel fluorescence in situ hybridization (FISH) technique called COMBinatorial Oligo FISH (COMBO-FISH). In contrast to other (standard) FISH applications, COMBO-FISH makes use of a bioinformatic approach for probe set design. By means of computer genome database search, oligonucleotide stretches of typical lengths of 15–30 nucleotides are selected in such a way that they all colocalize within a given genome (gene) target. Typically, probe sets of about 20–40 stretches are designed within 50–250 kb, which is enough to get an increased fluorescence signal specifically highlighting the target from the background. Although “specific colocalization” is the only necessary condition for probe selection, i.e. the probes of different lengths can be composed of purines and pyrimidines, we additionally refined the design strategy restricting the probe sets to homopurine or homopyrimidine oligonucleotides so that depending on the probe orientation either double (requiring denaturation of the target double strand) or triple (omitting denaturation of the target strand) strand bonding of the probes is possible. The probes used for the protocols described below are DNA or PNA oligonucleotides, which can be synthesized by established automatized techniques. We describe different protocols that were successfully applied to label gene targets via double- or triple-strand bonding in fixed lymphocyte cell cultures, bone marrow smears, and formalin-fixed, paraffin-wax embedded tissue sections. In addition, we present a procedure of probe microinjection in living cells resulting in specific labeling when microscopically detected after fixation.

Key words

COMBO-FISH Combinatorial oligo fluorescence in situ hybridization FISH Computer-based probe selection DNA/PNA oligonucleotides Lymphocyte hybridization Tissue hybridization Bone marrow smear hybridization Microinjection of oligonucleotides Microscopic detection of genome targets 

Notes

Acknowledgment

The financial support of the German Federal Minister of Education and Research (BMBF) is gratefully acknowledged (grant no. 01IG07015G (Services@MediGrid) and grant no. 13N8350).

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Eberhard Schmitt
    • 1
    • 2
  • Jutta Schwarz-Finsterle
    • 1
  • Stefan Stein
    • 1
  • Carmen Boxler
    • 1
  • Patrick Müller
    • 1
  • Andriy Mokhir
    • 3
  • Roland Krämer
    • 3
  • Christoph Cremer
    • 1
  • Michael Hausmann
    • 1
    Email author
  1. 1.Kirchhoff-Institute of PhysicsUniversity of HeidelbergHeidelbergGermany
  2. 2.Leibniz-Institute for Age ResearchJenaGermany
  3. 3.Institute of Inorganic ChemistryUniversity of HeidelbergHeidelbergGermany

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