A genomic library is a collection of the total genomic DNA from a single organism. The DNA is stored in a population of identical vectors, each containing a different insert of DNA. The organism's DNA is extracted from cells and digested with a restriction enzyme to cut the DNA into fragments of a specific size. The fragments are then inserted into the vector using DNA ligase. The vector DNA can be taken up by a host organism - commonly a population of Escherichia coli or yeast - with each cell containing only one vector molecule. Using a host cell to carry the vector allows for easy amplification and retrieval of specific clones from the library for analysis.
STEPS:
1. Isolating genomic DNA from the organism –
a) Isolation of DNA (purification) Eukaryotes: Prepare cell nuclei, remove proteins, lipids and other unwanted macromolecules by protease digestion and phase extraction.
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b) Prokaryotes: Extracted DNA directly from cells.
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2. Cutting of DNA into suitable fragment size: Genomic DNA is then partially cleaved into thousands of fragments (of 5-100 kb) by restriction endonuclease to get inserts of desired size range, compatible with the cloning vector (such as plasmid, phage lambda, cosmid, bacteriophage P1,Bacterial Artificial Chromosome [BAC] or Yeast Artificial Chromosome [YAC]) used for library construction. Cleaving the entire genome of a cell with a specific restriction nuclease and cloning each fragment is sometimes called the “shotgun” approach to gene cloning. This technique can produce a very large number of DNA fragments. Digestion into small fragments can be achieved by physical shearing (Pipetting, mixing). Restriction enzyme digestion : Partial digestion is preferred to get a greater lengths of DNA fragments.
Selection of restriction enzymes: 1. Ends produced (sticky or blunt) & the cleaved ends of the to be vector 2. Whether the enzyme is inhibited by DNA modifications 3. Time of digestion and ratio of restriction enzyme to DNA is dependent on the desired insert size range.
3. Incorporation of vector into suitable host: All the fragments are ligated into the cloning vector which is cleaved with the same restriction endonuclease and transformed into host organism which is commonly a population of Escherichia coli or yeast cells to produce a library with each cell containing one vector
molecule.
4. Maintenance of clones: Each cloning vector contains a different fragment of the genome so that all DNA in the genome is represented among the clones in the library. All of the plasmids in a particular host cell harbour the same insert. As the plasmid multiplies, there will be 50-100 copies of the plasmid in each cell. Genomic DNA inserts can be sequenced by isolating individual recombinant cloning vectors from cells. By sequencing and analysing random clones from the library configs can be made and the whole genome sequence can be recovered.
Application:
1. Determining the complete genome sequence of a given organism.
2. Serving as a source of genomic sequence for generation of transgenic animals through genetic engineering.
3. Study of the function of regulatory sequences in vitro.
4.
Study of genetic mutations in cancer tissues.
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| CONSTRUCTION OF GENOMIC LIBRARY |
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