disadvantages of plasmid vectors
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DNA Cloning with Plasmid Vectors
The essence of cell chemistry is to isolate a particular cellular component and then analyze its chemical structure and activity. In the case of DNA, this is feasible for relatively short molecules such as the genomes of small viruses. But genomes of even the simplest cells are much too large to directly analyze in detail at the molecular level. The problem is compounded for complex organisms. The human genome, for example, contains about 6 × 109 base pairs (bp) in the 23 pairs of chromosomes. Cleavage of human DNA with restriction enzymes that produce about one cut for every 3000 base pairs yields some 2 million fragments, far too many to separate from each other directly. This obstacle to obtaining pure DNA samples from large genomes has been overcome by recombinant DNA technology. With these methods virtually any gene can be purified, its sequence determined, and the functional regions of the sequence explored by altering it in planned ways and reintroducing the DNA into cells and into whole organisms.
The essence of recombinant DNA technology is the prep-aration of large numbers of identical DNA molecules. A DNA fragment of interest is linked through standard 3′ → 5′ phosphodiester bonds to a vector DNA molecule, which can replicate when introduced into a host cell. When a single recombinant DNA molecule, composed of a vector plus an inserted DNA fragment, is introduced into a host cell, the inserted DNA is reproduced along with the vector, producing large numbers of recombinant DNA molecules that include the fragment of DNA originally linked to the vector. Two types of vectors are most commonly used: E. coli plasmid vectors and bacteriophage λ vectors. Plasmid vectors replicate along with their host cells, while λ vectors replicate as lytic viruses, killing the host cell and packaging the DNA into virions (Chapter 6). In this section, the general procedure for cloning DNA fragments in E. coli plasmids is described.
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