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Related Concept Videos

In-situ Hybridization02:31

In-situ Hybridization

In situ hybridization (ISH) is a technique used to detect and localize specific DNA or RNA molecules in cells, tissue, or tissue sections using a labeled probe. The technique was first used in 1969 for the investigation of nucleic acids. It is currently an essential tool in scientific research and clinical settings, especially for diagnostic purposes.
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A probe is a complementary strand of DNA or RNA that binds to corresponding nucleotide sequences in a cell. Many...
Hybrid Zones02:29

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Hybrid zones are narrow regions where two closely related species interact, mate, and produce hybrids. Relative to either parent species, hybrids may possess distinct phenotypic or genetic differences that impact their survival and reproductive success. The genetic variances introduced by hybridization influence species diversity and speciation processes within the hybrid zone.Gene flow and natural selection are evolutionary mechanisms that shape the outcome of a hybrid zone. Gene flow...
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FISH - Fluorescent In-situ Hybridization

Fluorescence in situ hybridization, or FISH, was developed in the early 1980s and has quickly become one of the most widely used techniques in cytogenetics. Labeled probes are used to bind complementary DNA or RNA sequences on a chromosome or in a region within a cell. Earlier, the probes could only be obtained by cloning or reverse transcription of a DNA template. Currently, the probe oligonucleotides can be synthesized synthetically. Additionally, with the advancement of optical techniques,...
Diversity in Cell Signaling Responses01:22

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The physiological function of a cell and cellular communication are outcomes of a range of extrinsic signals, intracellular signaling pathways, and cellular responses. No two cell types express the same repertoire of signaling components. Receptors are highly selective for their cognate ligands, but once activated, they can alter multiple cellular processes such as DNA transcription, protein synthesis, and metabolic activity. 
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Before the start of mitosis and meiosis I, the cell synthesizes DNA, resulting in two homologous copies of each chromosome. DNA synthesis is...

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Heterokaryon Technique for Analysis of Cell Type-specific Localization
09:31

Heterokaryon Technique for Analysis of Cell Type-specific Localization

Published on: March 11, 2011

Hybridization kinetics is different inside cells.

Ingmar Schoen1, Hubert Krammer, Dieter Braun

  • 1Systems Biophysics, Center for Nanoscience, Ludwig Maximilians Universität München, Amalienstrasse 54, 80799 Munich, Germany.

Proceedings of the National Academy of Sciences of the United States of America
|December 19, 2009
PubMed
Summary
This summary is machine-generated.

Cellular reaction kinetics differ from bulk solutions. This study reveals DNA hybridization rates in living cells are modulated by binding interactions, not just crowding.

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Adaptation of Hybridization Capture of Chromatin-associated Proteins for Proteomics to Mammalian Cells

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Cell Biology

Background:

  • Cellular environments (macromolecular crowding, binding) may alter reaction kinetics compared to bulk solutions.
  • Understanding in vivo reaction kinetics is challenging due to difficulties in capturing fast cellular processes.
  • DNA hybridization kinetics in living cells remain largely elusive.

Purpose of the Study:

  • To spatially resolve and quantify DNA hybridization kinetics within single living cells.
  • To investigate the influence of cellular environment and DNA probe characteristics on reaction rates.
  • To develop and apply advanced imaging techniques for probing biomolecular interactions in vivo.

Main Methods:

  • Transfection of HeLa cells with Förster Resonance Energy Transfer (FRET)-labeled dsDNA probes.
  • Utilizing laser-driven temperature oscillations and stroboscopic fluorescence imaging for kinetic measurements.
  • Employing temperature oscillation optical lock-in microscopy to capture kinetics from 10 microseconds to 1 second.

Main Results:

  • DNA hybridization time constants varied with DNA concentration both within and between cells.
  • A 16-bp probe showed accelerated kinetics in cells compared to free solution, while a 12-bp probe exhibited decelerated kinetics.
  • In vitro experiments showed no significant effect of crowding agents on hybridization kinetics.

Conclusions:

  • Cellular reaction rates are specifically modulated by binding interactions, influenced by factors like DNA probe length.
  • The developed imaging modality enables probing of biomolecular interactions within cellular compartments for systems biology.
  • Findings highlight the distinct nature of in vivo kinetics compared to in vitro or bulk solution conditions.