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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). Gene targets can be specifically labelled with at least about 20 PNA-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. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants.more » • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.« less

Authors:
; ;  [1];  [1];  [2];  [1]
  1. University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg (Germany)
  2. (Germany)
Publication Date:
OSTI Identifier:
22648586
Resource Type:
Journal Article
Resource Relation:
Journal Name: Experimental Cell Research; Journal Volume: 345; Journal Issue: 1; Other Information: Copyright (c) 2016 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved.; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ARCHITECTURE; CELL NUCLEI; CHAINS; CHROMOSOMES; CLATHRATES; COMPUTERS; DETECTION; DIAGNOSIS; FLUORESCENCE; GENES; HEAT; HEAT TREATMENTS; INDICATORS; INTERACTIONS; LABELLING; LASERS; LIBRARIES; MAMMARY GLANDS; MICROSCOPY; NANOSTRUCTURES; NEOPLASMS; NUCLEI; OLIGONUCLEOTIDES; PEPTIDES; POLYCYCLIC AROMATIC HYDROCARBONS; POLYMERASE CHAIN REACTION; PROBES; SPECIFICITY; SYNTHESIS; THERAPY; VIABILITY

Citation Formats

Müller, Patrick, Rößler, Jens, Schwarz-Finsterle, Jutta, Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de, University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen, and Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei. United States: N. p., 2016. Web. doi:10.1016/J.YEXCR.2016.05.001.
Müller, Patrick, Rößler, Jens, Schwarz-Finsterle, Jutta, Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de, University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen, & Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de. PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei. United States. doi:10.1016/J.YEXCR.2016.05.001.
Müller, Patrick, Rößler, Jens, Schwarz-Finsterle, Jutta, Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de, University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen, and Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de. Fri . "PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei". United States. doi:10.1016/J.YEXCR.2016.05.001.
@article{osti_22648586,
title = {PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei},
author = {Müller, Patrick and Rößler, Jens and Schwarz-Finsterle, Jutta and Schmitt, Eberhard, E-mail: eschmitt@kip.uni-heidelberg.de and University of Göttingen, Institute for Numerical and Applied Mathematics, Lotzestraße 16-18, D-37083 Göttingen and Hausmann, Michael, E-mail: hausmann@kip.uni-heidelberg.de},
abstractNote = {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). Gene targets can be specifically labelled with at least about 20 PNA-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. - Highlights: • Denaturation free protocols preserve 3D architecture of chromosomes and nuclei. • Labelling sets are determined in silico for duplex and triplex binding. • Probes are produced chemically with freely chosen backbones and base variants. • Peptide nucleic acid backbones reduce hindering charge interactions. • Intercalating side chains stabilize binding of short oligonucleotides.},
doi = {10.1016/J.YEXCR.2016.05.001},
journal = {Experimental Cell Research},
number = 1,
volume = 345,
place = {United States},
year = {Fri Jul 01 00:00:00 EDT 2016},
month = {Fri Jul 01 00:00:00 EDT 2016}
}