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Home >> Recombinant DNA Technology >> Selection of Recombinant Lambda DNAs
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Selection of Recombinant Lambda DNAs - The recombinant to DNA can be selected in one of the following three ways.
1. Some A vectors, e.g., λgt10, retain the lysogenic, function so that they form turbid or cloudy plaques. In such vectors, the DNA fragment is inserted within the gene cl (lysis repressor) so that the recombinant DNA is cI-, and forms clear plaques, which are easily distinguishable from the cloudy plaques produced by the unaltered vector.
When such vectors are tested on an E. coli strain carrying the mutation hflA 150 (high frequency lysogeny), only the recombinant DNA form plaques (all the non-recombinant vector molecules enter the lysogenic cycle).

2. Some A vectors contain the promoter, operator and lacZ gene of E. coli. Insertion of DNA fragments in such vectors is accompanied with the deletion of a major part of the lacZ gene. Therefore, the unchanged DNA forms β-galactosidase, while the recombinant DNA does not.
When such a vector is tested on a lacZ- E. coli lawn grown on media having X-gal and IPTG, the unaltered vector produces blue plaque, while the recombinant DNA produces colourless plaque and is readily identifiable. This strategy is employed by the λgt11 vector; it is suited for all λ vectors that are > 38 kb in their unaltered state.
Both λgt10 and λgt11 vectors have been designed to clone cDNA. λgt11 is an expression vector, the protein encoded by the DNA insert being expressed as a β-galactosidase fusion protein. The DNA insert size for λgt10 and λgt11 must not exceed 7 and 6 kb, respectively.

3. In case of many A vectors, called replacement vectors, insertion of the DNA fragments is accompanied with the deletion of all or a major part of the nonessential segment specifying lysogeny. In general, such deleted vectors are smaller than 38 kb, the minimum genome size essential for packaging of the vector into phage heads.

The recombinant DNAs, on the other hand, are much larger than 38 kb and are packaged into phage particles. Therefore, packaging into phage heads after DNA insert integration offers an efficient selection strategy for recombinant DNAs. In this case, the vector and DNA insert sizes are carefully adjusted for an effective selection.

Lambda vectors EMBL3 and EMBL4, both used for preparing genomic libraries of eukaryotes, employ this strategy. These vectors easily accept DNA inserts in excess of 20 kb; they contain polylinkers having the reverse order, with respect to each other, of unique restriction sites.

 
 

 
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