Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Library Screening
DNA Probe Hybridization

Both Genomic Libraries and complementary DNA (cDNA) libraries contain hundreds of thousands or even millions (in the case of higher eukaryotes) of clones. Two fundamentally different approaches are used to identify a specific Gene or another DNA region.

The first detection method (screening) relies on The Use of oligonucleotide DNA probes that bind to the clones under study.

The second screening method is based on the expression of Proteins encoded by the given clone.

Let us examine the first method.

The screening method using oligonucleotide probes is based on Hybridization—the ability of complementary DNA or RNA molecules to specifically bind (hybridize) to one another through The formation of complementary nucleotide pairs.

The DNA double helix denatures (melts) upon heating (Figure 4) in a low-salt solution. If the Temperature is subsequently lowered and/or the ionic concentration in the solution is increased, the complementary single-stranded DNA molecules will reassociate (hybridize), reforming The Double Helix (renaturation of two DNA strands into a double-stranded molecule).

In a mixture of Nucleic Acids, only complementary single-stranded DNA molecules spontaneously renature; moreover, such renaturation is virtually independent of the presence of other, non-complementary strands in the solution.

Figure 92 shows a schematic diagram of membrane hybridization analysis.

First, the target DNA is denatured, and the single-stranded DNA molecules are irreversibly attached to a solid support—usually a nitrocellulose filter or nylon membrane.

Next, the filter is incubated in a solution containing radiolabeled single-stranded DNA probes. Under conditions favorable for hybridization (neutral pH; T=40–65°C; 0.3–0.6 M NaCl), the probes bind to the complementary DNA strands attached to the membrane. Unbound probes are washed off the filter, and the radiolabeled hybrids are detected by autoradiography. Autoradiography (or radioautoradiography) is a technique used to visualize the distribution of radioactive zones formed after electrophoretic Separation of a mixture of macromolecules extracted from a Cell, a tissue section, or a gel plate following the binding of radioactive probes to them.

Class="center">

Figure 92 – Detection of DNA by membrane hybridization analysis

To record the radioactive zones, an X-ray film is placed over the test sample, which is exposed precisely in the areas where the radioactive probes are concentrated.

Membrane hybridization analysis can be performed for both DNA and RNA, and the radiolabeled complementary probes must be either DNA or RNA, respectively.

The application of this λ-cDNA library screening Procedure is illustrated in Figure 93.

Figure 93 – Screening of a cDNA library using radioactive probes

A replica of the Petri dish containing A large number of λ-clones is first replicated onto The surface of a nitrocellulose membrane.

The membrane is then analyzed using radiolabeled probes specific for the recombinant DNA containing the target fragment.

Membrane hybridization with radiolabeled oligonucleotides is commonly used for screening λ-cDNA libraries. Once a cDNA clone encoding a specific protein is obtained, the full-length cDNA can be radiolabeled and used as a probe to identify clones containing fragments of the corresponding gene within a genomic library.