BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part I. General Biotechnology

CHAPTER 5. FUNDAMENTALS OF GENETIC ENGINEERING

5.1. BIOTECHNOLOGY OF RECOMBINANT DNA CONSTRUCTION

5.1.1. Preparation of DNA Fragments

Although studying the general biochemical properties of cellular DNA provided certain insights, uncovering the fine details of its genetic Organization remained impossible. By the mid-1970s, two widely adopted Methods significantly streamlined DNA analysis. One of these approaches relies on the discovery of hydrolytic Enzymes—Restriction Endonucleases (restriction enzymes)—which cleave DNA into fragments at specific nucleotide sequences present within the DNA molecule.

For molecular biology Applications, restriction enzymes are isolated from bacterial Cells. For instance, the restriction endonuclease EcoRI cleaves DNA exclusively at the GAATTC nucleotide sequence; SmaI hydrolyzes DNA at the CCCGGG nucleotide sequence, while the recognition site for BamHI is the GGATTC nucleotide combination. Other restriction enzymes target distinct nucleotide sequences. The specific sequences recognized and cleaved by these and other restriction endonucleases are distributed throughout the double-stranded Introduction/20.html">DNA Structure. Restriction enzymes make it possible to break down a DNA macromolecule into a set of fragments ranging from several hundred to several thousand Base Pairs in length. Fragments varying in molecular weight can be isolated using gel Electrophoresis, enabling subsequent analytical study of each purified fraction.

The second methodological breakthrough involves the relatively rapid determination of The nucleotide sequence of both restriction-generated DNA fragments and the macromolecule as a whole. However, this approach presents significant challenges because the total number of base pairs forming a DNA nucleotide sequence is excessively large, even within a bacterial Cell. As for the mammalian genome, which is vastly larger, it consists of approximately 2.5 billion base pairs that, in turn, form discrete functional blocks known as genes. Their total number in the mammalian genome reaches 50,000 to 100,000.

Evidence indicates that each Gene determines The structure of a specific protein. Consequently, researchers focused on investigating The nucleotide sequences of biological objects whose genomes are considerably smaller than Eukaryotic Cell genomes. Viruses served as ideal models. In 1979, researchers successfully determined the complete nucleotide sequence of the simian virus 40 (SV40) genome. It was established that this viral genome consists of 5,243 base pairs organized into five distinct genes. This model was particularly advantageous because the analysis of individual genes was not complicated by a large excess of unrelated sequences. Furthermore, viral Replication yields several hundred thousand copies of The Genome simultaneously within The Cell, greatly simplifying the Separation of viral DNA from host cell DNA. Because METABOLISM/28.html">The Genetic Code translating nucleotide sequences into Amino acid sequences had already been cracked, it became possible to deduce The amino acid sequences of the Proteins encoded by all five SV40 viral genes.

Simultaneously with elucidating the nucleotide sequence of the SV40 DNA molecule, researchers identified regions outside the structural genes—regions that do not encode proteins but instead participate in the Regulation of Gene Expression and Viral DNA replication.

Molecular biologists have developed methods for isolating genes from donor organisms, inserting these genes into vector molecules to generate recombinant (hybrid) DNA, ensuring the self-replication of recombinant DNA, transferring the hybrid DNA into a recipient Organism (host cell), and driving the expression of foreign genes.