GENERAL MICROBIOLOGY - T.P. Pyrog - 2004

19. BACTERIAL GENETICS: CONSTANCY, VARIATION, AND TRANSFER OF TRAITS

19.3. TRAIT TRANSFER AND GENETIC RECOMBINATION

19.3.7. General Principles of Gene Cloning (Construction of Hybrid DNA Molecules In Vitro)

Introduction/32.html">Genetic Engineering is a relatively young methodological field of molecular biology and biotechnology. The integration of independently developed Methods in the early 1970s made it possible to establish the modern strategy of genetic engineering, which essentially involves the following steps: 1) DNA fragments from any source are enzymatically inserted in vitro (in a test tube) into a DNA molecule capable of autonomous Replication independent of the host chromosome (a plasmid or viral DNA); 2) the resulting hybrid molecules are introduced into susceptible host Cells; 3) within these cells, the hybrid DNA molecules replicate, thereby amplifying the cloned DNA fragment; 4) specific Selection methods are used to isolate clones of cells or Viruses containing individual hybrid DNA molecules; 5) the identified hybrid DNAs are subjected to comprehensive Structural and functional analysis.

The simplest and most popular method for generating hybrid DNA molecules is the restriction-ligation method (Fig. 19.3).

The first hybrid molecules using this approach were obtained in 1973 by the American scientists S.N. Cohen and H.W. Boyer. Hybrid molecules are also referred to as recombinant molecules.

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Fig. 19.3. Generation of hybrid DNA by inserting eukaryotic DNA into a bacterial plasmid (simplified scheme)

The plasmid is cleaved by a restriction endonuclease (restriction enzyme) capable of generating single-stranded "sticky" (i.e., complementary) ends on the DNA fragment. One such restriction enzyme is EcoRI, for example. The same enzyme is used to fragment the DNA containing the Gene of interest. When these DNA fragments are mixed with the linearized plasmid, they have a certain probability of annealing via their complementary sticky ends. Subsequently, the foreign DNA is covalently ligated to the plasmid (vector) using the enzyme DNA ligase. The hybrid (recombinant) DNA molecule is then introduced into a host Cell via transformation, where it replicates along with the plasmid. The Cell progeny containing the hybrid DNA is genetically homogeneous, thus forming a clone.

Genetic engineering has unlocked unprecedented opportunities for creating fundamentally new producers of BIOLOGICALLY ACTIVE SUBSTANCES.