PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets

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Robust 3D DNA FISH Using Directly Labeled Probes
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Robust 3D DNA FISH Using Directly Labeled Probes

Published on: August 15, 2013

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Patrick Müller1, Jens Rößler1, Jutta Schwarz-Finsterle1

  • 1University of Heidelberg, Kirchhoff Institute for Physics, Im Neuenheimer Feld 227, D-69120 Heidelberg, Germany.

Experimental Cell Research
|May 31, 2016
PubMed

Insights

COMBinatorial Oligonucleotide FISH (COMBO-FISH) offers enhanced specificity and eliminates harsh DNA denaturation steps. This advanced technique preserves cellular nanostructure and viability for gene detection, aiding cancer diagnostics.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Established FISH probes, like BAC clones, often require harsh DNA denaturation steps.
  • PCR-constructed oligonucleotide FISH probes show improved targeting specificity but still rely on denaturing protocols.
  • Current labeling protocols involve heat treatment and chemical agents, potentially damaging cellular structures.

Purpose of the Study:

  • To introduce a novel FISH technique, COMBinatorial Oligonucleotide FISH (COMBO-FISH), for enhanced specificity and reduced sample processing.
  • To develop a method that omits DNA denaturing procedures, preserving cellular nanostructure and viability.
  • To demonstrate the application of COMBO-FISH for detecting key cancer-related genes.

Main Methods:

  • In silico design of specific oligonucleotide probe combinations for COMBO-FISH.
  • Synthesis of probes as single-stranded PNA-probes or TINA-DNA, independent of PCR or primer libraries.
  • Utilizing triplex-forming oligonucleotides to eliminate the need for DNA denaturation steps.

Main Results:

  • COMBO-FISH enables specific gene target labeling with high probe numbers (≥20), resulting in background-free specimens.
  • The elimination of denaturing steps successfully maintains the 3D nanostructure and viability of the target cells.
  • Successful detection of Her2/neu and GRB7 genes, crucial indicators in breast cancer diagnosis and therapy.

Conclusions:

  • COMBO-FISH represents a significant advancement in oligonucleotide-FISH technology.
  • The method offers a gentler, more specific, and efficient approach to gene detection.
  • COMBO-FISH has potential applications in diagnostics, particularly for cancer biomarkers, by preserving cellular integrity.
Keywords:
Chemical oligonucleotide synthesisCombinatorial sequence searchConfocal laser-scanning microscopyFluorescence DNA labellingHoogsteen triple strand bindingPeptide nucleic acids

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