COMBO-FISH Enables High Precision Localization Microscopy as a Prerequisite for Nanostructure Analysis of Genome LociReportar como inadecuado




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1

Kirchhoff-Institute for Physics, Im Neuenheimer Feld 227, 69120 Heidelberg, Germany

2

German Cancer Research Center DKFZ, Im Neuenheimer Feld 580, 69120 Heidelberg, Germany





*

Author to whom correspondence should be addressed.



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. View Full-Text

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 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





Autor: Patrick Müller 1, Eberhard Schmitt 1, Anette Jacob 2, Jörg Hoheisel 2, Rainer Kaufmann 1, Christoph Cremer 1 and Michael Hausmann 1,*

Fuente: http://mdpi.com/



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