Isolated DNA Fragments Can Be Cloned into E. coli Plasmid Vectors
FIGURE 6-13 Basic components of a plasmid cloning vector that can replicate within an E. coli cell. Plasmid vectors contain a selectable gene, such as ampr, which encodes the enzyme β-lactamase and confers resistance to ampicillin. Exogenous DNA can be inserted into the bracketed region without disturbing the ability of the plasmid to replicate or express the ampr gene. Plasmid vectors also contain a replication origin (ORI) sequence where DNA replication can be initiated by host-cell enzymes. Inclusion of a synthetic polylinker containing the recognition sequences for several different restriction enzymes increases the versatility of a plasmid vector. The vector is designed so that each site in the polylinker is unique on the plasmid.
Plasmids are circular, double-stranded DNA (dsDNA) molecules that replicate separately from a cell’s chromosomal DNA. These extrachromosomal DNAs, which occur naturally in bacteria and in lower eukaryotic cells (e.g., yeast), exist in a symbiotic relationship with their host cell. Like the host-cell chromosomal DNA, plasmid DNA is duplicated before every cell division. During cell division, copies of the plasmid DNA segregate to each daughter cell, ensuring continued propagation of the plasmid through successive generations of the host cell.
The plasmids most commonly used in recombinant DNA technology are those that replicate in E. coli. Investigators have engineered these plasmids to optimize their use as vectors in DNA cloning. For instance, removal of unneeded portions from naturally occurring E. coli plasmids yields plasmid vectors about 1.2–3 kb in circumferential length that contain three regions essential for DNA cloning: a replication origin (ORI); a marker that permits selection, usually a drug-resistance gene; and a region in which exogenous DNA fragments can be inserted (Figure 6-13).
Figure 6-14 outlines the general procedure for cloning a DNA fragment using E. coli plasmid vectors. When E. coli cells are mixed with recombinant vector DNA and subjected to a stress such as heat shock, a small fraction of the cells will take up the plasmid DNA, a process known as transformation. Typically, 1 cell in about 10,000 incorporates a single plasmid DNA molecule and thus becomes transformed. The rare transformed cells can be easily selected by use of a selectable marker. For instance, if the plasmid carries a gene that confers resistance to the antibiotic ampicillin, transformed cells can be selected by growing them in an ampicillin-containing medium. All the antibiotic-resistant progeny cells that arise from the initial transformed cell will contain plasmids with the same inserted DNA. Since all the cells in a colony arise from a single transformed parent cell, they constitute a clone of cells, and the initial fragment of DNA inserted into the parental plasmid is referred to as cloned DNA or a DNA clone.
EXPERIMENTAL FIGURE 6-14 DNA cloning in a plasmid vector permits amplification of a DNA fragment. A fragment of DNA to be cloned is first inserted into a plasmid vector containing an ampicillin-resistance gene (ampr), such as that shown in Figure 6-13. Only the few cells transformed by incorporation of a plasmid will survive on ampicillin-containing medium. In transformed cells, the plasmid DNA replicates and segregates into daughter cells, resulting in the formation of an ampicillin-resistant colony in which each cell contains the cloned DNA.
The versatility of an E. coli plasmid vector is increased by the addition of a polylinker, a synthetically generated sequence containing one copy of each of several different restriction sites that are not present elsewhere in the plasmid sequence (see Figure 6-13). Typically, the polylinker is cleaved with two different restriction enzymes in preparation to accept a DNA fragment prepared with two different sticky ends corresponding to the same two enzymes. Such a strategy eliminates unwanted by-products such as reclosing of the cleaved plasmid vector and greatly increases the efficiency of cloning DNA fragments.
Plasmid cloning vectors are useful for propagating DNA fragments up to about 20 kb in length, but fragments longer than this cannot be reliably replicated within one cell-division cycle. For some purposes, such as the isolation and manipulation of large segments of the human genome, it is desirable to clone DNA segments as large as several megabases [1 megabase (Mb) = 1 million base pairs]. For this purpose, specialized plasmid vectors known as BACs (bacterial artificial chromosomes) have been developed. One type of BAC uses a replication origin derived from an endogenous plasmid of E. coli known as the F factor. The F factor, and cloning vectors derived from it, can be stably maintained at a single copy per E. coli cell even when they contain inserted sequences of up to about 2 Mb. Production of BAC libraries requires special methods for the isolation, ligation, and transformation of large segments of DNA because segments of DNA larger than about 20 kb are highly vulnerable to mechanical breakage even by standard manipulations such as pipetting.