DNA Libraries Can Be Screened by Hybridization to an Oligonucleotide Probe

Both genomic and cDNA libraries of various organisms contain hundreds of thousands to upwards of a million individual clones in the case of higher eukaryotes. Two general approaches are available for screening libraries to identify clones carrying a gene or other DNA region of interest: (1) detection with oligonucleotide probes that bind to the clone of interest and (2) detection based on expression of the encoded protein. Here we describe the first method; an example of the second method is presented in the next section.

The basis for screening with oligonucleotide probes is hybridization, the ability of complementary single-stranded DNA or RNA molecules to associate (hybridize) specifically with each other via base pairing. As discussed in Chapter 4, double-stranded (duplex) DNA can be denatured (melted) into single strands by heating in a dilute salt solution. If the temperature then is lowered and the ion concentration raised, complementary single strands will reassociate (hybridize) into duplexes. In a mixture of nucleic acids, only complementary single strands (or strands containing complementary regions) will reassociate; moreover, the extent of their reassociation is virtually unaffected by the presence of noncomplementary strands.

In the membrane-hybridization assay outlined in Figure 9-16, a single-stranded nucleic acid probe is used to detect those DNA fragments in a mixture that are complementary to the probe. The DNA sample first is denatured and the single strands attached to a solid support, commonly a nitrocellulose filter or treated nylon membrane. The membrane is then incubated in a solution containing a radioactively labeled probe. Under hybridization conditions (near neutral pH, 40-65 °C, 0.3-0.6 M NaCl), this labeled probe hybridizes to any complementary nucleic acid strands bound to

▲ EXPERIMENTAL FIGURE 9-16 Membrane-hybridization assay detects nucleic acids complementary to an oligonucleotide probe. This assay can be used to detect both DNA and RNA, and the radiolabeled complementary probe can be either DNA or RNA.

the membrane. Any excess probe that does not hybridize is washed away, and the labeled hybrids are detected by auto-radiography of the filter.

Application of this procedure for screening a \ cDNA library is depicted in Figure 9-17. In this case, a replica of the petri dish containing a large number of individual \ clones initially is reproduced on the surface of a nitrocellulose membrane. The membrane is then assayed using a radiolabeled probe specific for the recombinant DNA containing the fragment of interest. Membrane hybridization with radiolabeled oligonucleotides is most commonly used to screen \ cDNA libraries. Once a cDNA clone encoding a particular protein is obtained, the full-length cDNA can be radiolabeled and used to probe a genomic library for clones containing fragments of the corresponding gene.

▲ EXPERIMENTAL FIGURE 9-17 Phage cDNA libraries can be screened with a radiolabeled probe to identify a clone of interest. In the initial plating of a library, the X phage plaques are not allowed to develop to a visible size so that up to 50,000 recombinants can be analyzed on a single plate. The appearance of a spot on the autoradiogram indicates the presence of a recombinant X clone containing DNA complementary to the probe. The position of the spot on the autoradiogram is the mirror image of the position on the original petri dish of that particular clone. Aligning the autoradiogram with the original petri dish will locate the corresponding clone from which infectious phage particles can be recovered and replated at low density, resulting in well-separated plaques. Pure isolates eventually are obtained by repeating the hybridization assay.

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