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Home >> Recombinant DNA Technology >> Identification of Clones Having Recombinant DNAs
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Identification of Clones Having Recombinant DNAs - The second step consists of identification and isolation of those clones that are transformed by the recombinant DNAs from among those that contain the unaltered vector. This may be achieved in one of several ways listed below.

1. In case the vector has two selectable markers, e.g., pBR322, the DNA insert may be placed within one of these markers, say, ampT gene. The other marker, in this case, tetr, is used for elimination of the nontransformed cells. The transformed clones are then replicaplated on ampicillin containing medium.

The clones containing the recombinant DNAs will be sensitive to ampicillin due to inactivation of the gene ampT by insertion of the DNA fragment. Such clones are identified and isolated from the master plate.

2. Some vectors contain a gene, or sometimes only part of a gene, which complements a function missing in their host cells, e.g., gene lacZα in the pUC vectors, which complements such lacZ- E. coli strains in which lacZα is deleted. The same combination is used for some A. vectors and M13 phage vectors. In all such cases, the DNA insert is so placed that it disrupts the expression of lacZα.

Therefore, E. coli cells containing the recombinant DNA are deficient in β-galactosidase and produce white colonies or plaques on a medium containing X-gal and IPTG. On the other hand, tells having the unchanged vector produce active β -galactosidase and give rise to blue colonies or plaques on the same medium. This allows an easy identification of the clones containing the recombinant DNAs.

3. When the DNA insert codes for a gene product, which is defective in the auxotrophic host cells, a direct selection for the recombinant DNA is possible. The host cells are grown on a medium lacking the compound needed by the auxotrophic host; only those cells, which contain the recombinant DNA can grow and form colonies. Obviously, this approach is limited in application.

4. Similarly, selection by suppression of nonsense mutations present in the host also permits a direct selection for the recombinant DNA.

5. Some A. vectors retain the lysogenic function as well, e.g., λgt10. In such vectors, the DNA insert may be placed within the lysis repressor gene cI- so that the vector becomes cI. As a result, cells transfected by the recombinant DNA will give rise to clear plaques, whereas those infected by the unaltered vector will yield cloudy or turbid plaques. Thus the recombinant DNAs are readily identified and isolated.

6. Some vectors, e.g., A. replacement vectors and cosmids, are much shorter than the minimum genome length needed for their packaging within virus particles. In such cases, the length of DNA insert can be so adjusted as to allow the packaging of only the recombinant DNA. This provides an efficient selection strategy for recombinant DNA.

 
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