title: PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.
abstract: Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy.
authors.affiliation: University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.
authors.forename: Patrick
authors.initials: P
authors.lastname: Müller
authors.affiliation: University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.
authors.forename: Jens
authors.initials: J
authors.lastname: Rößler
authors.affiliation: University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.
authors.forename: Jutta
authors.initials: J
authors.lastname: Schwarz-Finsterle
authors.affiliation: University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany; University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen, Germany. Electronic address: eschmitt@kip.uni-heidelberg.de.
authors.forename: Eberhard
authors.initials: E
authors.lastname: Schmitt
authors.affiliation: University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany. Electronic address: hausmann@kip.uni-heidelberg.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Hausmann
mesh_terms: D002467:Cell Nucleus / Q000378:metabolism*; D002886:Chromosomes, Human, Pair 17 / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods*; D003198:Computer Simulation; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D008214:Lymphocytes / Q000378:metabolism; D015335:Molecular Probes / Q000378:metabolism*; D020135:Peptide Nucleic Acids / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: D015335:Molecular Probes; D020135:Peptide Nucleic Acids
keywords: Chemical oligonucleotide synthesis; Combinatorial sequence search; Confocal laser-scanning microscopy; Fluorescence DNA labelling; Hoogsteen triple strand binding; Peptide nucleic acids