Selection of COMBO-FISH Probes for Multi-Purpose Applications

Stuhlmüller, Michael; Hausmann, Michael

doi:10.4172/2376-130x.1000131

Cited by 3 publications

(4 citation statements)

References 19 publications

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“…In so-called low-temperature FISH experiments, thermal denaturation of the target DNA has been omitted and measurements with scanning near-field optical microscopy (SNOM) have shown persuasive results regarding the preservation of metaphase chromosome morphology by renouncement of denaturation [ 7 ]. Based on these low-temperature protocols, a novel technique called combinatorial oligonucleotide (COMBO)-FISH was established and proven by Hausmann et al [ 15 ] (for review, see [ 16 ]). Oligonucleotide sets with a length of 15 to 30 bases per probe are used [ 17 ] that specifically bind within a designated genomic region.…”

Section: Introductionmentioning

confidence: 99%

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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Immunostaining and fluorescence in situ hybridization (FISH) are well established methods for specific labelling of chromatin in the cell nucleus. COMBO-FISH (combinatorial oligonucleotide fluorescence in situ hybridization) is a FISH method using computer designed oligonucleotide probes specifically co-localizing at given target sites. In combination with super resolution microscopy which achieves spatial resolution far beyond the Abbe Limit, it allows new insights into the nano-scaled structure and organization of the chromatin of the nucleus. To avoid nano-structural changes of the chromatin, the COMBO-FISH labelling protocol was optimized omitting heat treatment for denaturation of the target. As an example, this protocol was applied to ALU elements—dispersed short stretches of DNA which appear in different kinds in large numbers in primate genomes. These ALU elements seem to be involved in gene regulation, genomic diversity, disease induction, DNA repair, etc. By computer search, we developed a unique COMBO-FISH probe which specifically binds to ALU consensus elements and combined this DNA–DNA labelling procedure with heterochromatin immunostainings in formaldehyde-fixed cell specimens. By localization microscopy, the chromatin network-like arrangements of ALU oligonucleotide repeats and heterochromatin antibody labelling sites were simultaneously visualized and quantified. This novel approach which simultaneously combines COMBO-FISH and immunostaining was applied to chromatin analysis on the nanoscale after low-linear-energy-transfer (LET) radiation exposure at different doses. Dose-correlated curves were obtained from the amount of ALU representing signals, and the chromatin re-arrangements during DNA repair after irradiation were quantitatively studied on the nano-scale. Beyond applications in radiation research, the labelling strategy of immunostaining and COMBO-FISH with localization microscopy will also offer new potentials for analyses of subcellular elements in combination with other specific chromatin targets.

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Section: Introductionmentioning

confidence: 99%

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Krufczik

Sievers

Hausmann

et al. 2017

IJMS

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“…COMBO-FISH (COMBinatorial oligo-nucleotide FISH [ 34 ]; for review of applications see [ 35 ]) can be seen as a significant step towards fulfilling the requirements of super-resolution localization microscopy. In contrast to FISH, using Watson–Crick binding bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or cosmid probes for instance, COMBO-FISH combines FISH with the design of highly-specific oligo-nucleotide probe sets for any given genome region in the human genome [ 36 , 37 ].…”

Section: Introductionmentioning

confidence: 99%

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

Hausmann

Ilić

Pilarczyk

et al. 2017

IJMS

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Understanding molecular interactions and regulatory mechanisms in tumor initiation, progression, and treatment response are key requirements towards advanced cancer diagnosis and novel treatment procedures in personalized medicine. Beyond decoding the gene expression, malfunctioning and cancer-related epigenetic pathways, investigations of the spatial receptor arrangements in membranes and genome organization in cell nuclei, on the nano-scale, contribute to elucidating complex molecular mechanisms in cells and tissues. By these means, the correlation between cell function and spatial organization of molecules or molecular complexes can be studied, with respect to carcinogenesis, tumor sensitivity or tumor resistance to anticancer therapies, like radiation or antibody treatment. Here, we present several new applications for bio-molecular nano-probes and super-resolution, laser fluorescence localization microscopy and their potential in life sciences, especially in biomedical and cancer research. By means of a tool-box of fluorescent antibodies, green fluorescent protein (GFP) tagging, or specific oligonucleotides, we present tumor relevant re-arrangements of Erb-receptors in membranes, spatial organization of Smad specific ubiquitin protein ligase 2 (Smurf2) in the cytosol, tumor cell characteristic heterochromatin organization, and molecular re-arrangements induced by radiation or antibody treatment. The main purpose of this article is to demonstrate how nano-scaled distance measurements between bio-molecules, tagged by appropriate nano-probes, can be applied to elucidate structures and conformations of molecular complexes which are characteristic of tumorigenesis and treatment responses. These applications open new avenues towards a better interpretation of the spatial organization and treatment responses of functionally relevant molecules, at the single cell level, in normal and cancer cells, offering new potentials for individualized medicine.

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“…Future attempts must also discover a method, equivalent to GFP-tagging of proteins, to label specific DNA loci in vivo. Combinatorial oligonucleotide fluorescence in situ hybridization (COMBO FISH), proposed by M. Hausmann (for review, see Burger et al (10)), might point the direction to this Holy Grail of cell biology (11).…”

mentioning

confidence: 99%

“…Evidently, SPDMs have strong potential to substantially participate in answering this and other fundamental questions associated with nanoparticle research: In addition to providing superresolution under physiological conditions, the method also enables spectroscopic resolution of nanoparticles of different sizes, as nanoparticles optically blink at a sizedependent wavelength (demonstrated for GNPs in Moser's work). Finally, Stuhlmüller and Hausmann (11) recently proposed new technology of how to deliver and retain therapeutically more efficient bigger gold nanoparticles in cells; GNPs were tagged with modified DNA oligonucleotides and conveyed into the cells by transfection. In Moser's work (1), the authors demonstrate, using SPDM, that this strategy allows enhanced and stable cellular internalization of these nanoparticles.…”

mentioning

confidence: 99%

Nanoscopy and Nanoparticles Hand-in-Hand to Fight Cancer: An Exciting Entrée into the Rising NANOworld

Falk

2016

Biophysical Journal

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Selection of COMBO-FISH Probes for Multi-Purpose Applications

Cited by 3 publications

References 19 publications

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Combining Low Temperature Fluorescence DNA-Hybridization, Immunostaining, and Super-Resolution Localization Microscopy for Nano-Structure Analysis of ALU Elements and Their Influence on Chromatin Structure

Challenges for Super-Resolution Localization Microscopy and Biomolecular Fluorescent Nano-Probing in Cancer Research

Nanoscopy and Nanoparticles Hand-in-Hand to Fight Cancer: An Exciting Entrée into the Rising NANOworld

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