Genomic library


A genomic library is a collection of clones, each containing multiple copies of an individual fragment of genomic DNA, that together comprise the entire genome of an organism. The procedure broadly involves isolating genomic DNA from an individual, fragmenting it and ligating into a suitable vector for cloning, then extracting the fragments and sequencing them to produce contigs (shotgun cloning).

THE PROCEDURE:

Prepare the insert genomic DNA – it is important to have pure, high-molecular weight DNA that is free of nicks – then enzymatically shear the DNA into fragments of a suitable size for ligation into a vector (20-25kb for lambda replacement vectors, or 40kb for cosmids; cosmids are usually preferable). Complete digestion with restriction enzymes produces genomic fragments that are too small for packaging; thus high-frequency partial digestion is more ideal. Note that high-frequency partial digestion also produces many overlapping fragments, which is necessary to produce contiguous sequences later on. Mechanical shearing of DNA is also possible, but less efficient as it produces blunt ends rather than sticky ends for vector ligation.

The fragments are purified to desired size for packaging using either gel electrophoresis or sucrose gradient centrifugation. The purified genomic DNA is ligated into either a cosmid or a phage whose arms have been purified from stuffer DNA - this will produce a recombinant concatemer which can be packaged into phage particles in vitro and used to infect E. Coli. Different plaques will contain different phages, each with a different fragment of genomic DNA.

ISOLATION OF GENOMIC DNA:

Before genomic DNA can be fragmented and ligated into a suitable cloning vector, it needs to be isolated from cells and purified. The method belows describes a simple protocol for this.

1. Break open the cells mechanically; the exact method for this depends on cell type. For instance, animal cells (which lack a cell wall) require only gentle lysis with non-ionic detergents to disrupt the membrane.

2. Remove proteins (‘deproteinise’) usually by digestion with proteinase K

3. Concentrate the nucleic acids using ethanol precipitation

a. Layer absolute ethanol solution over a solution of concentrated DNA (at a ratio of 2.5 volumes ethanol to 1 volume DNA solution, for less concentrated DNA)
b. Withdraw fibres of DNA with a glass rod
d. Collect precipitated DNA by centrifugation

4. Remove RNA, using RNase, then precipitate with ethanol again

5. Determine the concentration of DNA; A260 = 1.0 is equivalent to 50µg/ml double-stranded DNA present.

6. If necessary (rarely), purify the DNA in caesium-chloride gradient centrifugation