Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Construction and Analysis of Genomic Libraries
Gene Cloning Strategy

The term "cloning" derives from the word "clone," which refers to a genetically homogeneous population of Cells or Viruses derived via asexual reproduction. The simplest example of a clone is an isolated bacterial colony grown on a solid medium. When discussing Gene cloning, we mean the Replication of multiple copies of a gene within a vector molecule inside the cells of a clone (or within Viral Particles inside a plaque). Consequently, every Cell in the clone (or every viral particle within an isolated negative colony) will carry identical fragments of foreign DNA. This is a crucial methodological technique that forms the cornerstone of all Introduction/32.html">Genetic Engineering. Indeed, when isolating genes by cleaving an Organism's DNA and incorporating the resulting fragments into vectors, a highly complex mixture of molecules is formed. In this mixture, a single gene or its segment represents a minuscule fraction, making it impossible to study either its Structure or The properties of its product. However, upon introducing recombinant DNA (a vector with an insert) into host cells or viral particles, only a single type of hybrid DNA gains The ability to replicate within each cell (or viral particle). By isolating a clone or the contents of a plaque, one can incubate them to obtain an unlimited quantity of cloned genes and their products.

The previous section already outlined several schemes for cloning DNA fragments using various vectors (Figs. 20.5–20.7). Summarizing this material, it should be noted that A wide variety of vector-host systems are employed for gene cloning. Among these, systems utilizing E. coli as the recipient cells have become the most widespread. Recombinant DNA is introduced into host cells or viral particles through various Methods.

To introduce plasmid-based vectors into cells, researchers use the transformation of competent cells or a more efficient approach: protoplast transformation (Chapter 2). However, this method of genetic transfer is characterized by a relatively low frequency (approximately 1 out of every 500 to 10,000 DNA molecules participates in transformation). Therefore, selecting cells that have successfully taken up the vector requires special markers integrated into its structure. Antibiotic Resistance genes are most commonly used as such markers, allowing for the direct Selection of transformed Bacteria on a medium supplemented with the appropriate antibiotic.

Vectors constructed on The basis of viruses, as well as cosmids and phasmids, offer advantages over Plasmid Vectors because they can be packaged efficiently into viral capsids in vitro and subsequently used to infect susceptible cells. The infection method is significantly more efficient than transformation, with roughly every tenth DNA molecule becoming infectious. Furthermore, once inside the cells, cosmids and phasmids are able to maintain themselves in a manner similar to plasmid DNA.

Another challenge in the cloning Procedure involves selecting clones that have taken up recombinant DNA specifically, rather than merely empty vector molecules, which are invariably present in the mixture generated during vector insertion. To overcome this problem, previously described techniques are employed, such as insertional marker inactivation. A more advanced method for selecting bacterial or phage clones carrying vectors with inserts is complementation analysis using mutant β-galactosidase genes (see above). This method allows bacterial clones (or phage plaques) containing recombinant DNA to be distinguished by their color when plated on a medium of a specific composition. Another similar example is The Use of the mel gene for constructing plasmid vectors. This gene encodes the enzyme tyrosinase, which catalyzes The conversion of Tyrosine into melanin (resulting in dark-colored colonies). When DNA fragments are inserted into the coding region of the mel gene, it becomes inactivated, causing the transformed colonies to lose their pigmentation. Various other techniques also exist for rapidly screening transformed or infected clones to identify those that have successfully acquired recombinant DNA.