A glaring problem in most areas of biochemical research is obtaining sufficient amounts of the substance of interest. For example, a 10 L culture of E. coli grown to its maximum titer will only contain about 7 mg of DNA polymerase I, and many other proteins in much lesser amounts. Furthermore, only rarely can as much as half of any protein originally present in an organism be recovered in pure form. Eucaryotic proteins are even more difficult to obtain because tissue samples are usually only available in small quantities. With regards to the amount of DNA present, the 10 L E. coli culture would contain about 0.1mg of any 1000 bp length chromosomal DNA but its purification in the presence of the rest of the chromosomal DNA would be a very difficult task. These difficulties have been greatly reduced through the development of molecular cloning techniques. These methods, which are also referred to as genetic engineering and recombinant DNA technology, deserve much of the credit for the enormous progress in biochemistry and the dramatic rise of the biotechnology industry.
The main idea of molecular cloning is to insert a DNA segment of interest into an autonomously replicating DNA molecule, a so-called cloning vector, so that the DNA segment is replicated with the vector. Cloning such a chimeric vector in a suitable host organism such as E. coli or yeast results in the production of large amounts of the inserted DNA segment. If a cloned gene is flanked by the properly positioned control sequences for RNA and protein synthesis, the host may also produce large quantities of the mRNA and protein specified by that gene. The techniques of genetic engineering are discussed in detail.
Plasmid-based cloning vectors
Plasmids are circular DNA duplexes of 1 to 200 kb that contain the requisite genetic machinery, such as replication origin, to permit their autonomous propagation in a bacterial host or in yeast. Plasmids may be considered molecular parasites but in many instances they benefit their host by providing functions, such as resistance to antibiotics, that the host lacks. Indeed, the widespread and alarming appearance, since antibiotics came into use, of antibiotic-resistant pathogens is a result of the rapid proliferation among these organisms of plasmids containing genes that confer resistance to antibiotics. Some types of plasmids, which are present in one or a few copies per cell, replicate once per cell division as does the bacterial chromosome; their replication is said to be under stringent control. The plasmids used in molecular cloning, however, are under relaxed control; they are normally present in 10 to as many as 700 copies per cell. Moreover, protein synthesis in the bacterial host is inhibited, for example, by the antibiotic chloramphenicol, thereby preventing cell division. These plasmids continue to replicate until two or three thousand copies have accumulated per cell. The plasmids that have been constructed for molecular cloning are relatively small, replicate under relaxed control, carry genes specifying resistance to one or more antibiotics, and contain a number of conveniently located restriction endonuclease sites into which the DNA to be cloned may be inserted. Indeed, many plasmid vectors contain a strategically located short segment of DNA known as a polylinker that has been synthesized to contain a variety of restriction sites that are not present elsewhere in the plasmid. You can refer to your restriction map of the PVL1392 vector to visualize this.
The expression of a chimeric plasmid in a bacterial host was first demonstrated in 1973 by Herbert Boyer and Stanley Cohen. The host bacterium takes up a plasmid when the two are mixed together in a process that is greatly enhanced by the presence of divalent cations such as Ca2+ (which increase membrane permeability of DNA). An absorbed plasmid vector becomes permanently established in its bacterial host with an efficiency of around 0.1%.
Plasmid vectors cannot be used to clone DNAs of more than 10 kb. This is because the time required for plasmid replication increases with plasmid size. Hence intact plasmids with large unessential inserts are lost through the faster proliferation of plasmids that have eliminated these inserts by random deletions.
Creating of the recombinant plasmids containing the target DNA
A DNA to be cloned is, in many cases, obtained as a defined fragment through the application of restriction endonucleases. Recall that most restriction enzymes cleave duplex DNA at specific palindromic sequences so as to yield single-stranded sticky ends that are complementary to each other. Therefore, as Janet Mertz and Ron Davis first demonstrated in 1972, a restriction fragment may be inserted into a cut made in a cloning vector by the same restriction enzyme. The complimentary ends of the two DNAs specifically associate under annealing conditions and are covalently joined through action of the enzyme DNA ligase. An inherent advantage of this method is the ability to precisely excise the desired fragment through its restriction sites.