Construction and screening of genomic and cDNA libraries

Formation of a genomic library using recombinant DNA technique
Fig. 39.17. Formation of a genomic library using recombinant DNA technique.
In order to isolate one or more related genes from a genome, we like to prepare a mixture of clones each carrying DNA derived either from the genomic DNA or from cDNA (derived from the mRNA isolated from a specific metabolically active tissue of an organism). This mixture may contain thousands of clones, which when derived directly from the genomic DNA are collectively called a genomic library. Similarly when these clones are cDNA, they are collectively called a cDNA library. Construction and use of these libraries will be discussed in this section.

Genomic library by shotgun experiment
Cloning an entire genome in the form of a library of random genomic clones (without identifying them) is often called a shotgun experiment. In this experiment, genomic DNA is extracted, broken into fragments of reasonable size by a restriction endonuclease and then inserted into a cloning vector to generate a population of chimeric vectors (Fig. 39.17). A set of fragments cloned in this manner is called a genomic library. Once such a library is available, then clones can be perpetuated indefinitely in a plasmid vector and retrieved whenever needed for a variety of purposes, mainly for identification and isolation of a gene, whenever a specific probe is available.
Formation of a genomic library using recombinant DNA technique
Fig. 39.17. Formation of a genomic library using recombinant DNA technique.

Genomic libraries can be prepared by using a number of restriction endonucleases, one at a time, so that fragments of varying sizes having cuts at different places of the genome will be available. However this may lead to cuts at inconvenient places, including sites within a gene, so that fragments having complete genes will be difficult to obtain. In order to overcome this difficulty, we use the following strategy in the shotgun experiment : (i) We use restriction endonucleases, which have short (4bp) recognition sequences, so that such a sequence may be frequently distributed, (ii) Conditions are used which give only partial digests, so that a particular restriction site is only occasionally cleaved, and long fragments without having any breaks on recognition sites available within a gene can be easily obtained. This technique of shotgun experiment leads to the construction of a random genomic library, in which all fragments have same fragment ends thus helping in retrieval of a fragment from the vector with the help of the same enzyme.

The number of fragments representing every sequence of the genome increases with genome size. For instance, for a probability level of 99% that all sequences are present in our library of a species, we may need 1,500 cloned fragments in E. coli; 4,600 in yeast; 48,000 in Drosophila melanogaster and 8,00,000 in a mammal like human being. Libraries reaching these desired limits have been prepared in all these cases.