Genomic+Library+Construction+Using+Plasmids


 * Genomic Library Construction with a Plasmid **

A genomic library describes a collection of DNA fragments derived from the genome of an organism and cloned randomly into suitable cloning vectors (plasmids, phages). If plasmid cloning vectors are used for the construction of the library, this library essentially is a collection of host cells, each of which contains a plasmid with an inserted DNA fragment. Together the individual fragments in the collection of inserted molecules represent the entire genetic information contained in an organism. This is known as a representative library. If the library has been established by using fragmented cellular DNA of an organism the library is called a genomic library. The term genomic DNA clone then refers to an individual cell carrying a cloning vector with one of the cellular DNA fragments. Subgenomic libraries are obtained only if selected portions of the genome, for example fragments from distinct sorted chromosomes, are represented in the library. A characteristic of genomic libraries is the fact that the individual inserts still contain non-coding intron sequences. Such libraries are used for determining the genomic structures of genes. Here’s a simple little picture. Enjoy.



A suitable cloning vector (in this example a circular plasmid) is linearized by cleavage with a suitable restriction enzyme (1). The cellular DNA containing the gene of interest (red) is also cleaved into fragments with this enzyme (2). For various reasons the cellular DNA is cleaved in a way yielding N overlapping fragments with a uniform length of 20 kb. Linearized vector DNA and genomic DNA fragments are then ligated in vitro, yielding a population of N recircularized cloning vector molecules, each of which contains an insert of cellular genomic DNA (3). These molecules are introduced into suitable host cells (4). The collection of cloning vectors with inserts obtained at step 3 or the collection of cells obtained after step 4 are called a gene library. This library is called a representative library if the sum of the genomic DNA inserts found in the cloning vectors of all cells represents the entire cellular DNA. In principle libraries can be made also with cellular RNA as starting material. The resulting collection of host cells containing recombinant vectors is then called a cDNA library. The art of cloning is to find the one particular cell (marked red) which contains the cloning vector with the gene of interest. This process is called library screening. If this goal has been achieved the gene in question is said to have been cloned. 