The ABCs of Gene Cloning by Dominic W. S. Wong

Author:Dominic W. S. Wong
Language: eng
Format: epub, pdf
Publisher: Springer International Publishing, Cham

Gene cloning requires, as an initial step, isolation of a specific gene encoding the protein of interest. Locating and selecting a single gene among thousands of genes in a genome is not a simple task.

12.1 The Genomic Library

For prokaryotes, identification of a particular gene is usually made by first constructing a genomic library (Fig. 12.1). The total genomic DNA is isolated, purified, and digested with a restriction enzyme . The short DNA fragments are then cloned into a suitable vector, for example, bacteriophage λ type vectors or phagemids . The resulting recombinant λ DNAs are assembled into phage particles by in vitro packaging, with all the required phage proteins provided by λ phage mutants that cannot replicate (see Sect. 9.​1.​2). Now, we have a library of all genomic DNA in short fragments (usually average 15 kb) cloned into the λ vectors packaged into viable phage particles. It should note that plasmids are in some cases used for constructing genomic libraries, although the size of the library would be generally smaller.

Fig. 12.1.Construction of genomic and cDNA libraries

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