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Membrane Hybridization to Screen a Library

Screening Libraries using Membrane Hybridization

Under the conditions of temperature and ion concentration found in cells, DNA is maintained in a duplex (two-stranded) structure by the hydrogen bonds that form betwen the A and T bases and G and C bases in each strand. DNA duplexes can be denatured into single strands by heating them, usually in a dilute salt solution (e.g., 0.01 M NaCl), or by raising the pH above 11. If the temperature is lowered and the ion concentration in the solution is raised, or if the pH is lowered to neutrality, the A-T and G-C base pairs reform between complementary single strands.

 

This process goes by many names: renaturation, reassociation, hybridization, annealing. In a mixture of nucleic acids, only complementary single strands (or strands containing complementary regions) will reassociate; the extent of their reassociation is virtually unaffected by the presence of noncomplementary strands. Such molecular hybridization can take place between two complementary strands of either DNA or RNA, or between an RNA strand and a DNA strand. To utilize molecular hybridization in the detection of specific DNA clones, single stranded DNA from recombinant DNA molecules is attached to a solid support, commonly a nitrocellulose filter or treated nylon membrane. When a solution containing single0stranded nucleic acids is dried on such a membrane, the single strands become irreversibly bound to the solid support in a manner that leaves most of the bases available for hybridization to a complementary strand. Although the chemistry if this irreversible binding is not well understood, the procedure is very useful. The membrane is then incubated in a solution containing radioactively labeled single-stranded DNA (or RNA) that is complementary to some of the nucleic acid bound to the membrane.

Under hybridization conditions (near neutral pH, 40-65 C, 0.3-0.6 M NaCl), this labeled probe hybridizes to the complementary nucleic acid bound to the membrane. Any excess probe that does not hybridize is washed away, and the labeled hybrids are detected by autoradiography of the filter. The recombinant lamda virions present in plaques of a lawn of E. coli are transferred to a nylon membrane by placing the membrane on the surface of the petri dish. Many of the viral particles in each plaque absorb to the surface of the membrane, but many virions remain in the plaques on the surface of the nutrient agar in the petri dish containing a large number of individual lamda clones is reproduced on the surface of the membrane.

The original petri dish is refrigerated to store the collection of lamda clones. The membrane is then incubated in an alkaline solution, which disrupts the virions, releasing and denaturing the encapsulated DNA. The membrane is then dried, fixing the recombinant lamda DNA to the membrane's surface. Next, the membrane is incubated with a radiolabeled probe under hybridization conditions. Unhybridized probe is washed away, and the filter is subjected to autoradiography.

The appearence of a spot on the autoradiogram indicates the presence of a recombinant lamda clone containing DNA complementary to the probe. The position of the spot on the autoradiogram corresponds to the position on the original petri dish where that particular clone formed a plaque. Since the original petri dish still contains many infectioua virions in each plaque, viral particles from the identified clone can be recovered for replacing by aligning the autoradiogram and the petri dish and removing viral particles from the clone corresponding to the spot. A similar technique can be applied for screening a plasmid library in E.coli cells.

 

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