Concepts

DNA fragments can be inserted into cloning vectors, stable pieces of DNA that will replicate within a cell. Cloning vectors must have an origin of replication, one or more unique restriction sites, and selectable markers. Plasmids are commonly used as cloning vectors.

Bacteriophage vectors Bacteriophages offer a number of advantages as cloning vectors. The most widely used bac-teriophage vector is bacteriophage X, which infects E. coli. One of its chief advantages is the high efficiency with which it transfers DNA into bacteria cells. A second advantage is that about a third of the X genome is not essential for infection and reproduction; without these genes, a X particle will still faithfully inject its DNA into a bacterial cell and reproduce. These nonessential genes, which comprise about 15 kb, can be replaced by as much as about 23 kb of foreign DNA. A third advantage is that DNA will not be packaged into a X coat unless it is 40 to 50 kb long; so fragments of foreign DNA are not likely to be transferred by the vector unless they are inserted into the X genome, which ensures that the foreign DNA fragment will be replicated after it enters the cell.

The essential genes of the phage X genome are located in a cluster. Strains of phage X, called replacement vectors, have been engineered with unique EcoRI sites on either side of the nonessential genes (I Figure 18.10) so that, by using EcoRI, the nonessential genes can be removed. Foreign DNA cut with EcoRI will have sticky ends that are complementary to those on the ends of the essential X genes, to which it can be connected by ligase. The X chromosome possesses short, single-stranded ends called cos sites that are required for packaging X DNA into a phage head. The recombinant phage chromosomes can then be packaged into protein coats and added to E. coli. The phages inject their recombinant DNA into the cell, where it will be replicated. Only DNA fragments of the proper size and containing essential genes will be packaged into the phage coats, providing an automatic selection system for recombinant vectors.

Cosmid vectors Although only about 23 kb of DNA can be cloned in X vectors, DNA fragments as large as about 44 kb can be cloned in cosmids, which combine the properties of plasmids and phage vectors.

Cosmids are small plasmids that carry phage X cos sites; they can be packaged into viral coats and transferred to bacteria by viral infection. Because all viral genes except the cos sites are missing, a cosmid can carry more than twice as much foreign DNA as can a phage vector. Cosmid vectors

Eco RI

Nonessential genes

'Phage chromosome ffi Cleavage allows nonessential genes to be removed.

^ The left and right arms are mixed with foreign DNA also cut by EcoRI.

Complementary sticky ends of DNA anneal.

^ The recombinant phage chromosome is then packaged into X protein coats.

^ The left and right arms are mixed with foreign DNA also cut by EcoRI.

Eco Rl

Complementary sticky ends of DNA anneal.

^ The recombinant phage chromosome is then packaged into X protein coats.

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