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Labeling DNA Probes03:31

Labeling DNA Probes

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.
Radioisotopes, fluorophores, or small molecule binding partners like biotin or digoxigenin, are the most widely used reporter tags for labeling DNA probes. These labels can be attached to the probe DNA molecule via...

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Quantitative PCR of T7 Bacteriophage from Biopanning
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Homogenous M13 bacteriophage quantification assay using switchable lanthanide fluorescence probes.

Ari Lehmusvuori1, Julius Manninen, Tuomas Huovinen

  • 1Department of Biochemistry and Food Chemistry, Division of Biotechnology, University of Turku, Finland. artule@utu.fi

Biotechniques
|November 15, 2012
PubMed
Summary

Researchers developed a rapid bacteriophage quantification method using single-stranded DNA (ssDNA) probes. This technique allows for quick and accurate measurement of bacteriophage titers, aiding in virological research and phage display applications.

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Area of Science:

  • Biotechnology
  • Molecular Biology
  • Virology

Background:

  • Bacteriophage quantification is crucial for applications like phage display and understanding viral reproduction.
  • Existing methods for bacteriophage titering can be time-consuming and lack sensitivity.

Purpose of the Study:

  • To develop a rapid and reliable method for bacteriophage quantification.
  • To enable efficient monitoring of bacteriophage titers using a novel DNA-based detection system.

Main Methods:

  • Developed a bacteriophage quantification assay based on measuring phage single-stranded DNA (ssDNA).
  • Utilized switchable lanthanide chelate complementation probes for sensitive ssDNA detection.
  • Designed probes with a lanthanide ion carrier chelate and a light-absorbing antenna ligand.

Main Results:

  • The method quantifies bacteriophage ssDNA through the formation of a fluorescent lanthanide chelate complex upon probe hybridization.
  • Achieved a dynamic range of 10(9)-10(12) colony-forming units per milliliter (cfu/mL).
  • The assay provides bacteriophage titers within 20 minutes in a microtiter well format.

Conclusions:

  • The developed method offers a rapid and reliable tool for bacteriophage quantification.
  • This technique is suitable for titering filamentous bacteriophages in phage display.
  • The assay has potential applications in virological research for analyzing ssDNA virus reproduction.