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Genomic Library

A genomic library contains all sequences from an organism's genome and is constructed through several steps: isolation of target DNA, fragmentation, cloning into a suitable vector, and screening. The screening process involves hybridization techniques to identify specific DNA sequences using labeled probes. Successful identification of transformed cell colonies allows for the maintenance of cloned DNA of interest.

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Genomic Library

A genomic library contains all sequences from an organism's genome and is constructed through several steps: isolation of target DNA, fragmentation, cloning into a suitable vector, and screening. The screening process involves hybridization techniques to identify specific DNA sequences using labeled probes. Successful identification of transformed cell colonies allows for the maintenance of cloned DNA of interest.

Uploaded by

Gogul Ramnath
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Genomic library

• A genomic library contains all the sequences present in the genome of an


organism. In the construction of genomic libraries it is feasible to use vectors that
could accommodate large size of inserts.

Steps in Genomic Library Construction:

Construction of genomic library involves following steps:

(а) Isolation of target DNA:

• Genomic libraries can be constructed by isolation of complete DNA from bacteria,


virus, plants and animals. In eukaryotes, high molecular weight DNA is isolated
by CTAB or SDS methods

(b) Restriction Fragments:

• Fragmentation can be done by mechanical shearing or using suitable restriction


enzymes. Partial digestion is essential to procure proper size DNA fragments.
Therefore, treatment times and concentration of enzyme is very important for
desirable result.
(c) Cloning into the suitable vector

• The suitable vector to prepare the genomic library can be selected based on
size of the fragment of genomic DNA and carrying capacity of the vector

• In the case of fragment generated by restriction enzyme, vector can be


digested with the same enzyme and put for ligation to get clone
• The recombinant vectors and the insert combinations are grown in a
bacterial host cell (E. coli). They replicate their genome along with the
vector genome contained within them

(d) Screening of Genomic library:

• Genomic library can be screened for clones by hy­bridization with probe

Screening by Colony Hybridization:

• Principally this screening technique involves hybridization between labelled


DNA probe and a target DNA sequence. Therefore, a target gene sequence
can be accurately identified.

(i) The initial step in screening a library—a colony of host cells are plated onto
solid medium containing antibiotics. The presence of antibiotics in the medium
ensures growth of only transformed cells due to its antibiotic resistance gene in
their plasmid.
(ii) When a discrete colony is formed on the plate, it is then transferred onto
nitrocellu­lose or nylon membrane commonly referred as solid matrix. The
exact position of cell colony on the plate is maintained on the matrix.

(iii) Once the cells are attached to solid matrix (nitrocellulose paper) cells are
lysed, deproteinised and released DNA is denatured by alkaline reagent.

(iv) The labelled DNA probe is mixed in the next step, to facilitate hybridization
between target DNA and DNA probe. The unbound probe is washed off from
the matrix

(v) Matrix is then subjected to autoradiography to determine the location of cells


con­taining hybridized DNA

(vi) Development of dark spot on the X­ray film indicates the presence of target
DNA (gene). The dark spot on X­ray film corresponds to the cell colony on the
master plate. Once transformed cell colony is identified, it is then sub­cultured
and main­tained as it carries cloned DNA of interest.

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