Southern blotting and hybridization with probes can be used to locate a few specific fragments in a large pool of DNA

Cloning vectors A cloning vector is a stable, replicating DNA molecule to which a foreign DNA fragment can be attached for introduction into a cell. An effective cloning vector has three important characteristics (I Figure 18.6): (1) an origin of replication, which ensures that the vector is replicated within the cell; (2) selectable markers, which enable any cells containing the vector to be selected or identified; (3) one or more unique restriction sites into which a DNA fragment can be inserted. The restriction sites used for cloning must be unique; if a vector is cut at multiple recognition sites, generating several pieces of DNA, there will be no way to get the pieces back together in the correct order. Three types of cloning vectors are commonly used for cloning genes in bacteria: plasmids, bacteriophages, and cosmids.

Plasmid vectors Plasmids are circular DNA molecules that exist naturally in bacteria (see p. 000 in Chapter 15). They contain origins of replication and are therefore able to replicate independently of the bacterial chromosome. The plasmids typically used in cloning have been constructed from the larger, naturally occurring bacterial plasmids.

The pUC19 plasmid is a typical cloning vector (iFigure 18.7). It has an origin of replication and two selectable markers—an ampicillin-resistance gene and a lacZ gene. Ampicillin is an antibiotic that normally kills bacterial cells, but any bacterium that contains a pUC19 plasmid will be resistant to this antibiotic. The lacZ gene encodes the enzyme p-galactosidase, which normally cleaves lactose to produce glucose and galactose (see p. 000 in Chapter 16). The enzyme will also cleave a chemical called X-gal, producing a blue substance; when X-gal is placed in the medium, any bacterial colonies that contain intact pUC19 plasmids will turn blue and can be easily identified. (In these experiments, the bacterium's own p-galactosidase gene has been inactivated, and so only bacteria with the plasmid turn blue.) The pUC19 plasmid also possesses a number of different unique restrictions sites grouped together (a polylinker) that allow DNA fragments to be inserted into the plasmid.

The easiest method for inserting a foreign DNA fragment into a plasmid is to use restriction cloning, in which the foreign DNA and the plasmid are cut by the same restriction enzyme. Restriction cloning produces complementary sticky ends on the foreign DNA and the plasmid (iFigure 18.8a). The DNA and plasmid are then mixed together; some of the foreign DNA fragments will pair with the cut ends of the plasmid. DNA ligase seals the nicks in the sugar-phosphate backbone, creating a recombinant plasmid that contains the foreign DNA fragment.

Although simple, restriction cloning has several disadvantages. First, restriction cloning requires that a single restriction site in the plasmid matches sites on both ends of the foreign sequence to be cloned. If this arrangement of restriction sites is not available, this relatively straightfor-

| First, a cloning vector must contain an origin of replication recognized in the host cell so that it is replicated along with the DNA that it carries.

Unique restriction-enzyme cleavage sites

Bam HI Pst I I

^ Third, a cloning vector needs a single cleavage site for one or more restriction enzymes.


(orgin of replication)

Selectable marker

Eco RI

Eco RI


^ Second, it should carry selectable markers—traits that enable cells containing the vector to be selected or identified.

4 18.6 Three characteristics of an idealized cloning vector.

An origin of replication, one or more selectable markers, and one or more unique restriction sites.

Eco RI Kpn I Bam HI

Was this article helpful?

0 0

Post a comment