I. Definition of Colony Hybridization

Colony Hybridization is the screening of a library with a labeled probe (radioactive, bioluminescent, etc.) to identify a specific sequence of DNA, RNA, enzyme, protein, or antibody.

How is this technique used and why is it important?

According to Brian White at MIT:

Hybridization has two important features:

1. Hybridization reactions are specific, probes will bind only to sites that have complimentary sequences.

2. Hybridization reactions occur in the presence of large quantities of molecules that are similar but not identical to the site. This means a probe can find one molecule of target in a mixture of millions of related but non-complementary molecules.

This specificity allows one to find a specific sequence in a complex mixture full of similar sequences. Hybridization techniques also allows scientists to pick out the molecule of interest from very complex mixtures and study the molecule on its own.

II. Performing Colony Hybridization

The following steps refer to the above image.

0. Isolate and grow cultures in a suitable medium (agar).

1. Transfer a sample of the colonies onto a solid matrix such as a nitrocellulose or nylon membrane.

2. The cells on the membrane are lysed and the DNA is then denatured.

3. A labeled PROBE is added to the matrix and hybridization takes place.

4. The matrix is rinsed to remove the non-hybridized probe molecules.

5. For radioactive probes, one uses autoradiography and the matrix is placed on x-ray film.

6. The film is observed for black spots that correspond to colonies that hybridized with the probe.

7. Compare the x-ray film with the master plate to see which colonies had probe hybridization. These are the colonies that contained the specific sequence that actually hybridized with the probe.

8. Colonies on the master plate that have the desired sequence can then be subcultured if desired.

III. Pros and Cons

PROS:

• Allows you to pick one molecule of interest out of millions.
• Does not require the isolation of nucleic acids.
• It is possible to culture and identify microorganisms containing the hybridized sequence.

CONS:

• Time consuming, takes time for colonies to incubate and for the hybridization to show on the x-ray film.
• When identifying microoganisms, the procedure will only work if the organisms will grow and form a detectable colony.

IV.Sources of Information

Davis, G., Duerr, B., Jacobs, T. Simultaneous screening of colony blots with radioactive and non-isotopic probes. Bio Techniques 12 (5): 688-689

Riley, L. K., Caffrey, C. J. Identification of enterotoxigenic E.coli by colony hybridization with nonradioactive digoxigenin-labeled DNA probes. J.Clin. Microbiol. 28(6): 1465-1468

V. Related Links

Screening DNA Libraries by Hybridization with Oligonucleotide Probes

Oligonucleotide Prode for the Specific Detection of Microorganisms: Novel Approach to the Study of Gut Microflora