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 through asexual reproduction. A simple example of a clone is an isolated bacterial colony grown on a solid medium. Gene cloning involves replicating 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 fundamental methodological approach that underpins all of Introduction/32.html">Genetic Engineering. After all, when genes are obtained by digesting 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 accounts for a negligible fraction, making it impossible to study either its Structure or The properties of its product. However, when recombinant DNA (a vector with an insert) is introduced into 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 plaque contents, one can incubate them and yield 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 cells as recipients 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 either the transformation of competent cells or a more efficient approach—protoplast transformation (Chapter 2). However, this method of genetic transfer has a relatively low frequency (approximately 1 out of every 500 to 10,000 DNA molecules participates in transformation). Therefore, to select for cells that have successfully taken up the vector, the vector must carry special markers. Most commonly, these markers are Antibiotic Resistance genes, which allow for the direct Selection of transformed Bacteria on a medium containing the corresponding antibiotic.

Virus-based vectors, as well as cosmids and phasmids, offer advantages over Plasmid Vectors because they can be packaged into viral capsids in vitro with high efficiency and subsequently used to infect susceptible cells. The infection method is far more efficient than transformation, with every tenth DNA molecule becoming infectious. Furthermore, once inside the cells, cosmids and phasmids are capable of being maintained in a manner similar to plasmid DNA.

Another challenge in the cloning Procedure is selecting those clones that have taken up recombinant DNA specifically, rather than empty vector molecules, which are invariably present in the mixture obtained when DNA fragments are incorporated into vectors. To address this issue, previously described techniques are used, such as insertional inactivation of a marker. A more advanced method for selecting bacterial clones or phages containing vectors with inserts is Complementation analysis utilizing mutant β-galactosidase genes (see above). Applying this method makes it possible to distinguish bacterial clones (or phage plaques) containing recombinant DNA 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 determines The structure of 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, and the transformed colonies lose their pigmentation. There are also other techniques for rapidly screening transformed or infected clones to identify those that have successfully taken up the recombinant DNA.