method requires no specific restriction sites and has great versatility; its chief drawback is that it creates a number of undesired products.
A fourth method, and one commonly used today, is the use of linkers to add complementary ends to DNA molecules (IFigure 18.8c). Linkers are small, synthetic DNA fragments that contain one or more restriction sites. The foreign DNA of interest is cut by any restriction enzyme; if sticky ends are created, they are digested to produce blunt ends. The linkers are then attached to the blunt ends by T4 ligase and are then cut by a restriction enzyme, generating sticky ends that are complementary to sticky ends on the plasmid, which have been generated by using the same restriction enzyme to cut the plasmid. Mixing the plasmid and foreign DNA leads to the formation of recombinant DNA that can be stabilized by ligase. The great advantage of using linkers is that a particular restriction site can be added at almost any desired location; so any two pieces of DNA can be cut and joined.
Transformation When a gene has been placed inside a plasmid, the plasmid must be introduced into bacterial cells. This task is usually accomplished by transformation, which is the capacity of bacterial cells to take up DNA from the external environment (see Chapter 8). Some types of cells undergo transformation naturally; others must be treated chemically or physically before they will undergo transformation. Inside the cell, the plasmids replicate and multiply.
The use of selective markers Cells bearing recombinant plasmids can be detected by using the selectable markers on the plasmid. One type of selectable marker commonly used with plasmids is a copy of the lacZ gene (I Figure 18.9). The lacZ gene contains a series of unique restriction sites into which may be inserted a fragment of DNA to be cloned. In the absence of an inserted fragment, the lacZ gene is active and produces p-galactosidase. When foreign DNA is inserted into the restriction site, it disrupts the lacZ gene, and p-galactosidase is not produced. The plasmid also usually contains a second selectable marker, which may be a gene that confers resistance to an antibiotic such as ampicillin.
Bacteria that are lacZ" are transformed by the plasmids and plated on medium that contains ampicillin. Only cells that have been successfully transformed and contain a plas-mid with the ampicillin-resistance gene will survive and grow. Some of these cells will contain an intact plasmid, whereas others possess a recombinant plasmid. The medium also contains the chemical X-gal. Bacterial cells with an intact original plasmid—without an inserted fragment—have a functional lacZ gene and can synthesize p-galactosidase, which cleaves X-gal and turns the bacteria blue. Bacterial cells with a recombinant plasmid, however, have a p-galactosidase gene that is disrupted by the inserted DNA; they do not synthesize p-galactosidase and remain white. Thus, the color of
\ 18.9 The lacZ gene can be used to screen bacteria containing recombinant plasmids. A special plasmid carries a copy of the lacZ gene and an ampicillin resistance gene. (Photo: Cytographics/Visuals Unlimited.)
the colony allows quick determination of whether a recombinant or intact plasmid is present in the cell.
Plasmids make ideal cloning vectors but can hold only DNA less than about 15 kb in size. When large DNA fragments are inserted into a plasmid vector, the plasmid becomes unstable. Cloning DNA fragments that are longer than 15 kb requires the use of different cloning vectors.
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