Biochemistry and Molecular Biology - Belyasova N.A. 2002

Fundamentals of Genetic Engineering
Construction and Analysis of Genomic Libraries

Introduction/32.html">Genetic Engineering, as the foundation of modern biotechnology, emerged in the 1970s from within the field of molecular biology. By that time, the mechanisms of the core template-directed processes occurring in Prokaryotic Cells—Replication, METABOLISM/31.html">Transcription, and Translation—had been deciphered and successfully replicated in vitro. The Structure of The Genetic Code was defined, and the first Gene (Yeast Alanine tRNA) was synthesized. Researchers successfully isolated and studied The properties of several Enzymes that utilize DNA AS A substrate (restriction enzymes, ligases, DNA polymerases). Embryologists mastered the methodology of transplanting somatic Cell nuclei from animals (such as frogs) to replace removed haploid nuclei, making it possible to reproduce animals asexually—in other words, to clone them (obtain genetically identical organisms).

Thus, for the first time, there arose the hope of replacing specific genes (such as defective ones) in Germ Cells with functional ones—in other words, performing Gene Therapy. However, this required mastering the methodology for isolating genes, identifying them, amplifying them, stably cultivating them as part of autonomously replicating molecules, and determining the properties of their gene products. All these challenges are addressed by genetic engineering, which refers to a set of molecular-Genetic Methods that enable the targeted design of organisms through the manipulation of their hereditary apparatus.

Because microorganisms, particularly Bacteria, are among the simplest forms of life and are much better understood genetically than macroorganisms, the initial experiments on obtaining recombinant DNA were performed precisely on bacterial cells. Soon, another major prospect for applying the advances of genetic engineering became apparent: the design of highly productive microbial strains that act as producers of BIOLOGICALLY ACTIVE SUBSTANCES possessing a tailored set of properties.

This chapter describes the principal methods that allow for obtaining specific genes, incorporating them into vector molecules for cloning in recipient cells, identifying genes within Genomic Libraries, and determining nucleotide sequences in DNA. Familiarity with this methodology is essential for modern biotechnologists who leverage the Achievements of Genetic engineering to create producer organisms for desired compounds.

To engineer organisms with specified traits, one must possess a set of genes that determine the desired Functions (such as the utilization of specific substrates, the Biosynthesis and secretion of particular products, resistance to certain Physical and Chemical factors, the degradation of xenobiotics, etc.). Such genes can be isolated from any Organism exhibiting the corresponding properties, but this first requires generating and characterizing a genomic library of that organism. A genomic library is defined as a collection of bacterial or bacteriophage clones, each containing a single type of recombinant DNA, which collectively represent the entire genome of the organism under study, with its fragments distributed across separate clones.