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Labeling DNA Probes03:31

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DNA probes are fragments of DNA labeled with a reporter tag to enable their detection or purification. The resulting labeled DNA probes can then hybridize to target nucleic acid sequences through complementary base-pairing, and may be used to recover or identify these regions.
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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,...
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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
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Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
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A fluorescence microscope uses fluorescent chromophores called fluorochromes, which can absorb energy from a light source and then emit this energy as visible light. Fluorochromes include naturally fluorescent substances (such as chlorophylls) and fluorescent stains that are added to the specimen to create contrast. Dyes such as Texas red and FITC are examples of fluorochromes. Other examples include the nucleic acid dyes 4’,6’-diamidino-2-phenylindole (DAPI), and acridine orange.
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Photoluminescence offers a wide range of applications due to its inherent sensitivity and selectivity. This technique allows for both direct and indirect analyses of the analyte. Direct quantitative analysis is possible when the analyte exhibits a favorable quantum yield for fluorescence or phosphorescence. However, an indirect analysis may be feasible if the analyte is not fluorescent or phosphorescent, or if the quantum yield is unfavorable. Indirect methods include reacting the analyte with...
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Updated: Apr 21, 2026

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes
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Fluorescent-protein-based probes: general principles and practices.

Hui-Wang Ai1

  • 1Department of Chemistry, University of California Riverside, Riverside, CA, 92521, USA, huiwang.ai@ucr.edu.

Analytical and Bioanalytical Chemistry
|October 20, 2014
PubMed
Summary
This summary is machine-generated.

Genetically encoded fluorescent probes derived from fluorescent proteins can track cellular dynamics like pH and calcium. This review outlines general principles for developing these versatile biosensors for broader biological research applications.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Background:

  • Fluorescent proteins are crucial for developing genetically encoded probes.
  • These probes respond to various cellular dynamics, including pH, redox, calcium, enzyme activity, and membrane potential.
  • Existing fluorescent-protein-based probes, despite their diversity, share fundamental development principles.

Purpose of the Study:

  • To outline the general principles and strategies for developing fluorescent-protein-based probes.
  • To illustrate these principles with specific examples.
  • To encourage wider adoption of probe development in biological studies.

Main Methods:

  • Review of established principles in fluorescent-protein probe development.
  • Categorization of probe strategies based on underlying mechanisms.
  • Inclusion of illustrative examples for each principle.

Main Results:

  • Identification of core principles guiding the design of fluorescent-protein sensors.
  • Demonstration of how these principles can be applied to create probes for diverse cellular targets.
  • Highlighting the straightforward nature of these principles for adaptation.

Conclusions:

  • The development of fluorescent-protein-based probes relies on a few core, adaptable principles.
  • Wider application of these principles can democratize sensor development beyond specialized labs.
  • Enhanced genetically encoded sensors will significantly benefit biological research.