Submit Article |Resources | Link to us | Sitemap | Search | Language
Back to Home
Home >>Molecular Probes and Gene Libraries >> Genomic Library by Shotgun Experiment
Back to Home

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 vector molecules.

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, including identification and isolation of a gene, when a specific probe is available.

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 (4 bp) 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 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 parobability level of 99% that all sequences are present in our library of a species, we may need 1,500 cloned fragments for E. ,coli, 4,600 for yeast, 48,000 for Drosophila melanogaster and 8,00,000 for a mammal like human being. Libraries reaching these desired limits have been prepared in all these cases.

 
 

Submit Article |Resources | Link to us | Sitemap | Search | Language
English|Français|Español|Deutsch|Italiano|Portugues|Japanese|Korean|Sim-Chinese| Language