COMBinatorial Oligo FISH: directed labeling of specific genome domains in differentially fixed cell material and live cells.
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.
Schmitt
Eberhard
E
Kirchhoff-Institute of Physics, University of Heidelberg, Heidelberg, Germany.
Schwarz-Finsterle
Jutta
J
Stein
Stefan
S
Boxler
Carmen
C
Müller
Patrick
P
Mokhir
Andriy
A
Krämer
Roland
R
Cremer
Christoph
C
Hausmann
Michael
M
D001483:Base Sequence; D001854:Bone Marrow Cells / Q000166:cytology; D002460:Cell Line; D002470:Cell Survival; D030561:Databases, Nucleic Acid; D005557:Formaldehyde / Q000378:metabolism; D016678:Genome / Q000235:genetics*; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D016612:Paraffin Embedding; D013048:Specimen Handling; D016707:Tissue Fixation
D005557:Formaldehyde