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Genomic Library - A genomic library is a collection of plasmid clones or phage lysates containing recombinant DNA molecules so that the sum total of DNA inserts in this collection, ideally, represents the entire genome of the concerned organism. However, inspite of all the care taken in the production of genomic libraries.

Certain DNA fragments should be expected to be under or over represented or even missing. There are several possible reasons for this, and at present they can not be taken care of.

Construction of A Genomic Library
For preparation of a genomic library, the total genomic DNA of an organism is extracted. The DNA is broken into fragments of appropriate size either by mechanical shearing (this generates blunt ended fragments), sonication, or by using a suitable restriction endonuclease for partial digestion of the DNA. Complete digestion is avoided since it generates fragments that are too heterogeneous in size.

For partial digestion, restriction enzymes having 4-base (tetrameric) recognition sequences are employed in preference to those having 6-base (hexameric) target sites.

This is because a given 4-base recognition site is expected to' occur every 44 (= 256) base pairs in a DNA molecule, while a 6 base target site would occur only after every 46 (= 4096) base pairs. (It is assumed here that the arrangement of the 4 bases in DNA molecules is random).

Therefore, the fragments produced in partial digests with enzymes having 4 base recognition sites are more likely to be of appropriate size for cloning than those generated by enzymes having 6 base recognition sites.

Single or mixed digestions with the enzymes AluI, HaeIII or Sau3A have been used for constructing genomic libraries. The use of restriction enzymes has the advantage that the same set of fragments are obtained from a DNA each time a specific enzyme is used, and many of the enzymes; produce cohesive ends.

The partial digests of genomic DNA are subjected to agarose gel electrophoresis or sucrose gradient centrifugation for separation from the mixture of fragment of appropriate size. These fragments are then inserted into a suitable vector for cloning.

This constitutes the shotgun approach to gene cloning. In principle, any vector can be used, but A vectors and cosmids have been the most commonly used since DNA inserts of upto 23-25 kb (kilobase pairs) can be cloned in these vectors. The vectors containing the inserts are cloned in a suitable bacterial host.

Identification of the Desired Clone
Identification of the bacterial colony containing the desired DNA fragments from among those making up the library.

 
   
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