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Related Experiment Video

Updated: Jul 7, 2026

Robust 3D DNA FISH Using Directly Labeled Probes

Robust 3D DNA FISH Using Directly Labeled Probes

Published on: August 15, 2013

Using DNA fragments as probes.

¹Harvard Medical School, Boston, Massachusetts, USA.

Current Protocols in Molecular Biology

|February 12, 2008

Summary

This study details a method for detecting specific DNA sequences in bacteriophage plaques or bacterial colonies using radioactive probes and filter hybridization. The protocol optimizes DNA reannealing near the melting temperature (Tm) for accurate clone identification.

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

Nucleic acid hybridization is fundamental to molecular biology techniques.
Efficient detection of specific DNA sequences in biological samples is crucial for research and diagnostics.
Optimizing hybridization conditions, such as temperature near the melting temperature (Tm), enhances specificity and sensitivity.

Purpose of the Study:

To describe a detailed procedure for detecting bacteriophage plaques and bacterial colonies using DNA hybridization.
To enable the identification of specific clones through hybridization with radioactive probes.
To present an alternative protocol that omits formamide from the hybridization solution.

Main Methods:

Bacteriophage plaques or bacterial colonies are immobilized on a filter membrane.

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Filters are prewet and placed in a sealed bag for hybridization with a radioactive DNA probe.

Post-hybridization washes remove excess probe, followed by autoradiography for clone visualization.

Main Results:

The described hybridization method successfully identifies clones containing specific DNA sequences.
Autoradiography allows for the precise localization of positive signals on the filter membrane.
An alternate protocol provides a variation in hybridization solution composition.

Conclusions:

This hybridization technique provides a reliable method for screening and identifying specific DNA sequences in microbial colonies.
The protocol is adaptable, with an alternative method offered for different experimental needs.
The method facilitates the discovery and characterization of desired clones in molecular biology research.