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Selection of Clone Containing A Specific DNA Insert -Once we obtain a population of recombinant clones the next step is to identify a clone, which has the DNA insert of interest. The technique used for identification has to be highly precise and extremely sensitive to allow an accurate detection of a single clone from among the thousands obtained from a cloning experiment. The various strategies used for the purpose are briefly outlined below.
Colony Hybridization. The most efficient and rapid strategy for identification of a clone having the desired insert uses the technique of colony hybridization. The bacterial colonies are replicaplated or phage plaques are directly lifted on nitrocellulose filters, the cells are lysed and their DNA is denatured, the filter is incubated with the specific radioactive 32p-labelled) probe under anealing conditions.
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After some time, the probe is washed out leaving only those probe molecules that have hybridized with the denatured DNA from bacterial cells or phage particles. The colonies/plaques with whose DNA the probe has hybridized are identified by autoradiography; these contain the desired DNA insert.
These colonies/plaques are isolated from the master plate used for replica plating.A very large number of colonies or plaques (upto 10,000 plaques) can be lifted on to a single 10 cm diameter filter. But it is essential that a specific probe for the DNA insert is available.
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A probe is a polynucleotide (DNA or RNA; usually small molecules of as few as 15 bases, but more often of 2530 bases) molecule of a specific base sequence, which is used to detect DNA molecules having the same base sequence by complementary base pairing.
Generally, the probes are labelled with 32p to enable autoradiography for an easy identification of the DNA samples that base-pair with the probe. It is desirable that the probes are single-stranded to avoid pairing between the two strands of the probe itself. Either DNA or RNA can be used as probe. There are several approaches for developing specific probes.
Other Approaches. When specific probes are not available, many indirect approaches may be used for the identification of clones having the desired DNA insert. These procedures are not generally convenient for screening of a large number of clones. Two of such procedures, called
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(1) hybrid arrested translation (HART) and
(2) hybrid selection, use in vitro translation systems and then identification of the resulting polypeptide(s).It is, therefore, necessary that the protein product of the DNA insert being searched should be known, at least in terms of its electrophoretic mobility.
Complementation. The cloned DNA insert may express itself in the bacterial cells; this is possible for prokaryotic genes, some yeast genes and for eukaryotic cDNAs cloned in suitable expression vectors. Eukaryotic sequences isolated from genomic DNA have to be expressed in appropriate eukaryotic hosts, e.g., yeast cells, animal cells m culture, etc.
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If the protein produced by the desired DNA insert is deficient in the host cells, this insert will correct the deficiency of the cells transformed by it, i.e., will complement the deficiency of host cells.
This can be stated in general terms as follows. The host cells are deficient in a protein A, i.e., they are A-. These cells can be used to isolate the DNA fragment coding for protein A from a mixture of DNA fragments. Expression of recombinant DNAs are prepared from the DNA fragments and A- host cells are transformed; these cells are now cultured under selective conditions that require functional A product.
Only those host cells that contain the DNA insert encoding protein A will be able to multiply under the selective conditions (since the DNA insert will provide functional protein A). This strategy is limited in application by the availability of appropriate host cells.
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Unique Gene Products. Alternatively, the protein product of DNA insert can be identified by its unique function, i.e., a function not performed by the proteins of nontransformed host cells. Such functions may relate to enzyme activities or hormone effects for which appropriate assays exist.
Antibodies Specific to the Protein Product. Finally, if the protein lacks a recognizable and measurable function, it can be detected by using specific antibodies. A practical approach is to divide the large number of recombinant clones into a convenient number of groups and to assay for the presence of the protein. The positive group is again divided into subgroups and assayed.
In this manner, the positive groups are subdivided again and again till a single positive clone is identified. This approach is applicable to the previous strategy as well. The identification of proteins using antibodies may be achieved by western blotting, precipitation and electrophoresis or ELISA (enzyme-linked immunosorbent assay; Appendix-2.IX).
Colony/Plaque Screening with Antibodies. An efficient and rapid screening using antibodies is as follows. The antibody specific to the concerned gene product (i.e., protein) is spread uniformly over a solid support, e.g., plastic or paper disc, which is placed in contact with an agar layer containing lysed bacterial colonies or phage plaques.
If any clone is producing the protein in question, it will bind to the antibody molecules present on the disc. The disc is removed from the agar, is treated with a second radiolabelled (generally with L25I) antibody, which is also specific to the same protein but in a region different from that recognised by the first antibody.
These antibodies, therefore, will also bind to the protein moleculed held by the first antibody; the location of radioactivity on the disc is determined by autoradiography. The colonies/plaques producing the protein are then identified and isolated from the master plate.
This technique is analogous to colony hybridization and is able to screen large numbers of clones rather rapidly. But for this technique we require two different antibodies, which bind to two distinct domains of the desired protein, and this protein must not be produced by the nontransformed host cells.
FACS. In case of animal cells, an automated system, called fluorescence activated cell sorter (FACS), can be used for very rapid (upto 1,000 cells/sec) sorting of transformed cells. This is applicable to all the genes whose products become arranged on the cell surface and are available for binding of specific antibodies.
Therefore, these proteins must not be produced by the nontransformed host cells. The antibody molecules are attached to a fluorescent molecule and the transformed cells are treated with this antibody specific for the desired protein. The cells containing on their surface the protein in question will interact with the fluorescent antibodies.
Cells are then passed one by one in a stream between a laser and a fluorescence detector. The cells which fluoresce are deflected into a microculture tray, while the nonfluorescing cells are drawn away by an aspirator.
This approach is also applicable to the genes encoding receptor proteins present on the cell surface; in such cases, fluorescent ligands (the concerned molecule to which the receptor binds) are used in the place of fluorescent antibodies.
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