Genomic Library

Genomic Library:

Genomic library, produced when the complete genome of a particular organism is cleaved into thousands of fragment and all the fragments are cloned by insertion into a cloning vector.

• The first step in preparing a genomic library is isolation and digestion of the DNA by restriction endonucleases - This process produces fragments of DNA that include the organism’s entire genome.
• A plasmid, BAC, YAC or bacteriophage vector is digested with the same enzyme.
• DNA ligase is used to ligate genomic DNA pieces and vector.

In theory, all DNA fragments in the genome will be cloned into a vector:

• Recombinant vectors are then used to transform bacteria or yeast cells.
• Each transformed bacterium or yeast cell grows into a colony, or “clone,” of identical.
• Cells, each cell-bearing the same recombinant plasmid.
• Consider each clone a “book “in this “library” of DNA fragments.

Making of Gene Libraries:

There are three general ways to produce genomic libraries:

1. Complete digestion with restriction enzyme, cleave at all relevant restriction sites. This has drawback:

• Genes containing one or more sites for the restriction enzyme will be cloned into two or more pieces.
• To screen the entire genome, a very large number of clones would have to be examined, because insert DNA size is relative small.

2. Longer DNA fragments can be generated with mechanical sheering (e.g. passage through a syringe needle) rather than restriction enzyme cutting. A disadvantage is the absence of uniform ends, require enzymatic modification before insertion into a cloning vector.

3. Partial digestion with a restriction enzyme is controlled so that it cuts only some of the available sites. Ideally, this results in cloning a population of overlapping fragments representing the entire genome.

i. Partially digested DNA molecules in a certain size range are selected by density gradient centrifugation or agarose gel electrophoresis.

ii. DNA fragments with sticky ends from restriction digestion can be cloned directly.

iii. Genomic sequences are not equally represented in the libraries, because:

• Regions of DNA with relevant restriction sites very close together or very far apart axe removed at the size selection.
• Some regions of eukaryotic DNA prevent vector replication in E coli, and so are eliminated from the library.

One disadvantage of creating this type of library for eukaryotic genes:

• Is that non-protein coding pieces of DNA called introns are cloned in addition to protein coding sequences (exons), because a majority of DNA in any eukaryotic organism consists of introns.
• Many of the clones in a genomic library will contain non-protein coding pieces of DNA.
• Another limitation of genomic libraries is that many organisms, including humans, have such a large genome that searching for a gene of interest really is like searching for a needle in a haystack!