Screening a Library by Hybridization

After cloning all the possible genes from an organism into a library, the next step is to identify the gene of interest. Libraries are often screened for the gene of interest by DNA/DNA hybridization using a DNA probe. The probes themselves are generally derived from two sources. Cloned DNA from a related organism is often used to screen a library. The stringency of the hybridization conditions must be adjusted to allow for a greater or lesser percentage of mismatches, depending on how closely related the two organisms are. Another possibility is to synthesize an artificial probe, using the base sequence deduced from the amino acid sequence of the corresponding protein. This assumes that the protein has been purified and that a partial amino acid sequence from the N-terminal region is available. The DNA probe is labeled for detection by autoradiography, fluorescence or chemical tagging as described in Ch. 21. Probes generally range from 100 to 1000 bases long, although shorter probes may sometimes be used. At least 80% matching over a 50 base stretch is needed for acceptable hybridization and identification.

The target DNA (i.e. the DNA from the library to be probed) is denatured and bound to a nitrocellulose or nylon membrane. The membrane is then incubated with the labeled probe. After washing away excess probe, the membrane is screened by the chosen detection system, e.g. autoradiography as illustrated in Fig. 22.24.

Gene libraries may also be screened for the proteins expressed from the genes, rather than the DNA sequences.

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