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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. 2016. "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 = 2016,
month = 7
}
  • Highlights: •Identified stomach lineage specific gene set (SLSGS) was found to be under expressed in gastric tumors. •Elevated expression of SLSGS in gastric tumor is a molecular predictor of metabolic type gastric cancer. •In silico pathway scanning identified estrogen-α signaling is a putative regulator of SLSGS in gastric cancer. •Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. -- Abstract: Stomach lineage specific gene products act as a protective barrier in the normal stomach and their expression maintains the normal physiological processes, cellular integrity and morphology of the gastric wall. However,more » the regulation of stomach lineage specific genes in gastric cancer (GC) is far less clear. In the present study, we sought to investigate the role and regulation of stomach lineage specific gene set (SLSGS) in GC. SLSGS was identified by comparing the mRNA expression profiles of normal stomach tissue with other organ tissue. The obtained SLSGS was found to be under expressed in gastric tumors. Functional annotation analysis revealed that the SLSGS was enriched for digestive function and gastric epithelial maintenance. Employing a single sample prediction method across GC mRNA expression profiles identified the under expression of SLSGS in proliferative type and invasive type gastric tumors compared to the metabolic type gastric tumors. Integrative pathway activation prediction analysis revealed a close association between estrogen-α signaling and SLSGS expression pattern in GC. Elevated expression of SLSGS in GC is associated with an overall increase in the survival of GC patients. In conclusion, our results highlight that estrogen mediated regulation of SLSGS in gastric tumor is a molecular predictor of metabolic type GC and prognostic factor in GC.« less
  • An integrated infinity-focused fiber optic probe designed for cone penetrometer and hand-held use and constructed at Lawrence Livermore National Laboratories is described and evaluated for performance. Throughput efficiency is calculated, distance dependence, background levels, and sensitivity to heterogeneity in samples is evaluated. Representative spectra are presented that make use of the major performance advantages of this probe, which are (a) background rejection, (b) low sample light fluences, (c) distant viewing, (d) sample penetration, and (e) sample averaging capability. [copyright] [ital 1999 American Institute of Physics.]
  • Induction of thymidine kinase parallels the onset of DNA synthesis. To investigate the transcriptional regulation of the thymidine kinase gene, the authors have examined whether specific nuclear factors interact in a cell-cycle-dependent manner with sequences upstream of this gene. Two inverted CCAAT boxes near the transcriptional initiation sites were observed to form complexes with nuclear DNA-binding proteins. The nature of the complexes changes dramatically as the cells approach DNA synthesis and correlates well with the previously reported transcriptional increase of the thymidine kinase gene.
  • Two isoforms of metallothionein (MT) have been isolated from rainbow trout livers following CdCl/sub 2/ injections. These MTs have been identified by standard procedures and appear to be similar to mammalian MTs. Total RNA from such induced livers was shown to contain high levels of MT-mRNA activity when translated in cell free systems. This activity was demonstrated to be in the 8 to 10S region of a sucrose gradient. The RNA fractions also showed homology to a mouse MT-I cDNA probe. The exposure of rainbow trout hepatoma (RTH) cells to various concentrations of CdCl/sub 2/ and ZnCl/sub 2/ induced themore » expression of MT and MT-mRNA. Exposure of Chinook salmon embryonic (CHSE) cells to these metals, however, did not result in MT synthesis, suggesting that the MT genes have not become committed to transcription. Instead, an unknown low molecular weight (MW = 14 kDa) protein was induced. This metal-inducible protein (MIP) was capable of binding /sup 109/Cd and was stable to heating, while the binding of the metal to this protein was not. These characteristics have been reported for a protein induced in rainbow trout liver following environmental exposure to cadmium.« less