BIOTECHNOLOGY - V. H. Gerasymenko - 2006

Part I. General Biotechnology

CHAPTER 5. FUNDAMENTALS OF GENETIC ENGINEERING

5.1. BIOTECHNOLOGY OF RECOMBINANT DNA CONSTRUCTION

5.1.3. Construction of Recombinant DNA

The Development of Recombinant DNA technology is one of the most remarkable achievements of molecular biology. DNA molecules, including giant eukaryotic DNA molecules carrying genes of interest to researchers, are cleaved into individual fragments using Restriction Endonucleases (restriction Enzymes). It is noteworthy that the same restriction enzyme is used to cleave both the DNA molecule from which the desired Gene is to be obtained and the vector DNA (a plasmid or temperate phage). In this case, a restriction endonuclease, such as EcoRI (a bacterial enzyme), cleaves short palindromic sequences (having a 2nd-order Symmetry axis) at specific sites within these sequences, cutting obliquely across both strands of the chromosomal DNA and the vector DNA. This generates complementary single-stranded overhangs known as sticky ends. Crucially, enzymes are successfully used to linearize both the chromosomal DNA—yielding numerous fragments—and the plasmid DNA. Subsequently, the single-stranded complementary ends of the large DNA molecule fragment and the plasmid undergo annealing, which leads to the joining

of sticky ends through nitrogenous base pairing, while any nicks present in The Structure of the annealed DNA are repaired using the readily available bacterial enzyme DNA ligase (Fig. 5.1).

Other Methods applied in Introduction/32.html">Genetic Engineering are also known (such as the homopolymeric tailing and linker methods), which enable the joining of unrelated DNA molecules. Joining two unrelated DNA molecules via the tailing method is achieved by the enzymatic addition of poly(dA) fragments to both 3'-ends of one of the two unrelated DNA molecules using terminal transferase (deoxynucleotidyl transferase); simultaneously, poly(dT) fragments are attached to both 3'-ends of the second DNA molecule. Alternatively, homopolymeric nucleotide sequences consisting of guanine and cytosine NUCLEOTIDES can be used to join the 3'-ends of a double-stranded DNA Structure. A distinctive feature of terminal deoxynucleotidyl transferase is that its action is independent of a polynucleotide template; therefore, the elongation of the 3'-ends of the joining DNA molecules can proceed using homopolymeric nucleotide sequences approximately 100 residues in length. Because the identical polynucleotide sequences attached to the 3'-ends may vary in length, any resulting defects in the double-helical DNA structure are filled in by DNA polymerase I.

Class="center">

Fig. 5.1. Scheme of recombinant DNA construction

(after D. Hopwood, 1984)

The joining of DNA and vector fragments generated by the action of appropriate restriction enzymes is carried out using a method that incorporates elements of both the tailing and sticky-end techniques. To achieve this, an oligonucleotide fragment consisting of 6-10 nucleotide Base Pairs is first chemically synthesized. This fragment serves a connecting function and is therefore called a linker. It is used to connect the ends of the cloned DNA fragment and the vector into which this fragment is inserted to form recombinant DNA molecules. A prerequisite for creating a linker is the possibility of its subsequent Cleavage by a corresponding restriction endonuclease. Next, the pre-synthesized linker (connecting chain) is covalently attached to the 3'-ends of the vector (whether plasmid-derived or otherwise) and the DNA fragments. Meanwhile, the 5'-ends of the linker, the cloned DNA region, and the vector DNA are phosphorylated using polynucleotide kinase and joined using a ligase (T4 phage), which forms a covalent bond between blunt DNA ends. Blunt ends—formed by attaching a linker to the cloned DNA fragment or vector after enzymatic Treatment with an appropriate restriction enzyme, such as EcoRI—are thus converted into sticky ends (Fig. 5.2), the joining of which is based on the mechanism described above.

Fig. 5.2. Scheme of joining DNA fragments by the combined (linker) method. Arrows indicate restriction enzyme cleavage sites

resulting in The formation of sticky ends

(after L. Stryer, 1985)

At the stage of isolating cloned genes, the total DNA of a donor Cell (e.g., a mammal) is cleaved into fragments ranging from 1 to 30 kb in size using restriction enzymes, which act as a kind of defense system protecting The Cell from foreign DNA invasion. The complex genome is successfully split into several hundred thousand polynucleotide fragments, each of which can be incorporated into a vector DNA molecule. It is crucial that the inserted polynucleotide fragment of the recipient cell's DNA does not exceed the polynucleotide capacity of the vector being used.

Recombinant (hybrid) DNA molecules are obtained from the fragments generated by endonuclease cleavage of total genomic DNA and vector DNA (plasmid or bacteriophage) using the sticky-end, tailing, or combined (linker) methods in the presence of DNA ligase. In the presence of all required components, The process of recombinant DNA formation is completed within a few minutes.

The next stage in recombinant DNA Cloning involves the introduction of these hybrid molecules—consisting of cloned DNA fragments (including those from eukaryotic organisms) embedded in vector DNA—into a bacterial host cell. The penetration of such 'naked' DNA molecules into Bacterial Cells and their ability to confer new hereditary traits upon these cells were described by American researchers from the laboratory of O.T. Avery (1944). The phenomenon of DNA uptake from the environment by bacterial and Eukaryotic cells was named transformation. The efficiency of transformation is very low: only about one in a million DNA molecules enters the cell. However, by establishing specific conditions, the number of transformed cells can be significantly increased.

An essential condition for obtaining recombinant DNA molecules is the Selection of Plasmids that contain only a single site for specific interaction with a given restriction endonuclease. Under this condition, circular plasmid DNA is converted into a single linear molecule upon enzymatic cleavage. If a plasmid contains multiple palindromic sequences specifically recognized by a restriction enzyme, the hydrolytic action of the enzyme causes the plasmid DNA to break down into several fragments. To obtain recombinant DNA molecules, it is necessary to engineer small plasmids characterized by a high Replication rate.

Today, when designing new vectors, the goal is already set at the planning stage to obtain recombinant molecules that, while possessing other necessary properties, can relatively easily penetrate the host cell. These requirements are met by a vector constructed from a mutant form of phage λ, designated as λ g t-λ. Upon treatment with the restriction enzyme EcoRI, this mutant is cleaved at two sites (whereas the wild-type has up to five cleavage sites). An important feature that allows this phage mutant to be used as a vector is that large regions of its DNA can be deleted without altering the infectious Properties of the bacteriophage. The DNA remaining after EcoRI cleavage accounts for 72% of the viral genome length. The defect created by the elimination of the viral DNA segment can be filled by a 10 kb DNA fragment intended for cloning. In this case, the hybrid DNA molecule—constructed on The basis of phage λ and retaining 93% of its original length (prior to restriction enzyme treatment)—can be encapsidated (packaged) into the viral HEAD. One of the main advantages of vectors created on the basis of phage λ is their ability to infect Bacteria with an efficiency significantly exceeding that of hybrid molecules constructed on a plasmid basis.