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COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci
Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany
German Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, 69120 Heidelberg, Germany
* Author to whom correspondence should be addressed.
Received: 15 September 2010; in revised form: 16 October 2010 / Accepted: 18 October 2010 / Published: 21 October 2010
(This article belongs to the Special Issue Biomarkers
Abstract: With the completeness of genome databases, it has become possible to develop a novel FISH (Fluorescence in Situ Hybridization) technique called COMBO-FISH (COMBinatorial Oligo FISH). In contrast to other FISH techniques, COMBO-FISH makes use of a bioinformatics approach for probe set design. By means of computer genome database searching, several oligonucleotide stretches of typical lengths of 15–30 nucleotides are selected in such a way that all uniquely colocalize at the given genome target. The probes applied here were Peptide Nucleic Acids (PNAs)—synthetic DNA analogues with a neutral backbone—which were synthesized under high purity conditions. For a probe repetitively highlighted in centromere 9, PNAs labeled with different dyes were tested, among which Alexa 488Ò showed reversible photobleaching (blinking between dark and bright state) a prerequisite for the application of SPDM (Spectral Precision Distance/Position Determination Microscopy) a novel technique of high resolution fluorescence localization microscopy. Although COMBO-FISH labeled cell nuclei under SPDM conditions sometimes revealed fluorescent background, the specific locus was clearly discriminated by the signal intensity and the resulting localization accuracy in the range of 10–20 nm for a detected oligonucleotide stretch. The results indicate that COMBO-FISH probes with blinking dyes are well suited for SPDM, which will open new perspectives on molecular nanostructural analysis of the genome.
Keywords: COMBO-FISH; combinatorial oligo fluorescence in situ hybridization, computer based probe selection; PNA; SPDM; spectral precision distance/position determination microscopy; localization microscopy; nanostructure analysis of the genome
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MDPI and ACS Style
Müller, P.; Schmitt, E.; Jacob, A.; Hoheisel, J.; Kaufmann, R.; Cremer, C.; Hausmann, M. COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci. Int. J. Mol. Sci. 2010, 11, 4094-4105.
Müller P, Schmitt E, Jacob A, Hoheisel J, Kaufmann R, Cremer C, Hausmann M. COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci. International Journal of Molecular Sciences. 2010; 11(10):4094-4105.
Müller, Patrick; Schmitt, Eberhard; Jacob, Anette; Hoheisel, Jörg; Kaufmann, Rainer; Cremer, Christoph; Hausmann, Michael. 2010. "COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome Loci." Int. J. Mol. Sci. 11, no. 10: 4094-4105.