Construction of Genomic Library : The process of subdividing genomic DNA into clonable elements and inserting them into host cells is called creating a library.
A complete library, by definition, contains the entire genomic DNA of the source organism and is called as genomic library. A genomic library is a set of cloned fragments of genomic DNA. The process of creating a genomic library includes four steps :
In the first step the high molecular weight genomic DNA is separated and subjected to restriction enzy me digestion by using two compatible restriction enzymes.
In the second step, the fragments are then fractionated or separated by using agarose gel electrophoresis to obtain fragments of required size.
These fragments are then subjected to alkaline phosphatase treatment to remove the phosphate. In the third step, the dephosphorylated insert is ligated into vector which could be a plasmid, phage or cosmid, depending upon the interest of the researcher.
In the last step, the recombinant vector is introduced into the host by electroporation and amplified in host. In principle, all the DNA from the source organism is inserted into the host but this is not fully possible as some DNA sequences escape the cloning procedure. Genomic library is a source of genes and DNA sequences. A genomic library is a set of cloned fragments of genomic DNA. Prior information about the genome is not required for library construction for most organisms. In principle, the genomic DNA, after the isolation, is subjected to RE enzyme for digestion to generate inserts.
cDNA libraries V/S Genomic libraries:
• Genomic library is a mixture of fragments of genomic DNA while cDNA obtained from mRNA may cloned to give rise to a cDNA library.Genomic library’ contains DNA fragments that represent genes as well as those that are not genes. In contrast cDNA library contains only those genes that are expressed in the concerned tissue/organism. In both cases, a mixture of fragments is used for cloning to establish the library.
• Use of cDNA is absolutely essential when the expression of an eukaryotic gene is required in a prokaryote.
• Eukaryotic cDNAs are free from intron sequences.
• As a result of the above, they are smaller in size than the corresponding genes, i.e., the genes that encoded them.
• A comparison of the cDNA sequence with the corresponding genome sequence permits the delineation of intron/exon boundaries.
• The contents of cDNA libraries from a single organism will vary widely depending on the developmental stage and the cell type used for preparation of the library. In contrast, the genomic libraries will remain essentially the same irrespective of the developmental stage and the cell type used.
• A cDNA library will be enriched for abundant mRNAs, but may contain only a few or no clones representing rare mRNAs.