TY  - JOUR
AU  - Krufczik, Matthias
AU  - Lee, Jin-Ho
AU  - Tchetgna, Florence Laure Djiki
AU  - Cremer, Christoph
AU  - Bestvater, Felix
AU  - Schmitt, Eberhard
AU  - Hausmann, Michael
PY  - 2019
DA  - 2019/02/19
TI  - COMBO-FISH: A Versatile Tool Beyond Standard FISH to Study Chromatin Organization by Fluorescence Light Microscopy
JO  - OBM Genetics
SP  - 064
VL  - 03
IS  - 01
AB  - Background: Fluorescence In Situ Hybridization (FISH) has become routine for bio-medical research and medical diagnosis, thereby offering a variety of probes and ready-to-use kits that fulfil requirements for many applications. However, conventional FISH relies on chemo- and/or thermal denaturation to improve target accessibility and use huge amounts of DNAs that needs to be bonded to the target site. COMBinatorial Oligo-nucleotide FISH (COMBO-FISH) offers possibilities to circumvent these shortcomings. 
Methods: COMBO-FISH uses either a set of oligo-nucleotide probes (15 – 25 mers) uniquely co-localizing at the target site or single oligo-stretches repetitively but exclusively binding to the target. These are designed by systematic sequence data base searches. COMBO-FISH probes form Hoogsteen or Watson-Crick bonds and protocols with or without thermal denaturation can be realized. The latter allows the combination of COMBO-FISH with immunostaining. Low amounts of probe DNA allow the best maintenance of native chromatin organization – a prerequisite for applying super-resolution single molecule localization microscopy (SMLM).
Results: Specific labelling of the AMACR gene with an oligo-nucleotide probe set was applied in three different cell types. Hybridization efficiencies were determined by counting spots of high visibility and their radial positions were measured by confocal microscopy. The nuclear architecture revealed a non-random organization. Using uniquely, repetitively binding COMBO-FISH probes for centromere 9, Alu consensus and L1 sequences revealed a characteristic probe distribution in cell nuclei as being measured by SMLM. Comparison to theoretical data allowed determination of chromosome 9 radial positions without painting whole chromosomes. Three-colour staining by COMBO-FISH for Alu and L1 with immunostaining for heterochromatin is successfully demonstrated. 
Conclusions: COMBO-FISH is a powerful tool that circumvents shortcomings of standard FISH procedures using probes derived e.g., from BAC clones. The application for measurements of nuclear architecture by 3D confocal microscopy and of chromatin nano-architecture by SMLM using COMBO-FISH and immunostaining simultaneously has been demonstrated.
SN  - 2577-5790
UR  - https://doi.org/10.21926/obm.genet.1901064
DO  - 10.21926/obm.genet.1901064
ID  - Krufczik2019
ER  -