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Cloning vector (plasmid)

@ Cloning vector is cleaved with restriction endonuclease.

Eukaryotic chromosome

Eukaryotic chromosome

DNA fragment of interest is obtained by cleaving chromosome with a restriction endonuclease.

© Fragments are ligated to the prepared cloning vector.

DNA ligase

Recombinant vector

© DNA is introduced into the host cell.

(5 Propagation (cloning) produces many copies of recombinant DNA.

(5 Propagation (cloning) produces many copies of recombinant DNA.

Schematic Illustration Dna Cloning

FIGURE 9-1 Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

FIGURE 9-1 Schematic illustration of DNA cloning. A cloning vector and eukaryotic chromosomes are separately cleaved with the same restriction endonuclease. The fragments to be cloned are then ligated to the cloning vector. The resulting recombinant DNA (only one recombinant vector is shown here) is introduced into a host cell where it can be propagated (cloned). Note that this drawing is not to scale: the size of the E. coli chromosome relative to that of a typical cloning vector (such as a plasmid) is much greater than depicted here.

by its own restriction endonuclease is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase. The restriction endonuclease and the corresponding methylase are sometimes referred to as a restriction-modification system.

There are three types of restriction endonucleases, designated I, II, and III. Types I and III are generally large, multisubunit complexes containing both the endonucle ase and methylase activities. Type I restriction endonu-cleases cleave DNA at random sites that can be more than 1,000 base pairs (bp) from the recognition sequence. Type III restriction endonucleases cleave the DNA about 25 bp from the recognition sequence. Both types move along the DNA in a reaction that requires the energy of ATP. Type II restriction endonucleases, first isolated by Hamilton Smith in 1970, are simpler, require no ATP, and cleave the DNA within the recognition sequence itself. The extraordinary utility of this group of restriction endonucleases was demonstrated by Daniel Nathans, who first used them to develop novel methods for mapping and analyzing genes and genomes.

Thousands of restriction endonucleases have been discovered in different bacterial species, and more than 100 different DNA sequences are recognized by one or more of these enzymes. The recognition sequences are usually 4 to 6 bp long and palindromic (see Fig. 8-20). Table 9-2 lists sequences recognized by a few type II restriction endonucleases. In some cases, the interaction between a restriction endonuclease and its target sequence has been elucidated in exquisite molecular detail; for example, Figure 9-2 shows the complex of the type II restriction endonuclease EcoRV and its target sequence.

Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to four nu-cleotides of one strand unpaired at each resulting end. These unpaired strands are referred to as sticky ends (Fig. 9-3a), because they can base-pair with each other or with complementary sticky ends of other DNA fragments. Other restriction endonucleases cleave both strands of DNA at the opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends (Fig. 9-3b).

The average size of the DNA fragments produced by cleaving genomic DNA with a restriction endonuclease depends on the frequency with which a particular restriction site occurs in the DNA molecule; this in turn depends largely on the size of the recognition sequence. In a DNA molecule with a random sequence in which all four nucleotides were equally abundant, a 6 bp sequence recognized by a restriction endonuclease such as BamHI would occur on average once every 46 (4,096) bp, assuming the DNA had a 50% GmC content. Enzymes that recognize a 4 bp sequence would produce smaller DNA fragments from a random-sequence DNA molecule; a recognition sequence of this size would be expected to occur about once every 44 (256) bp. In natural DNA molecules, particular recognition sequences tend to occur less frequently than this because nucleotide sequences in DNA are not random and the four nucleotides are not equally abundant. In laboratory experiments, the average size of the fragments produced by restriction en-donuclease cleavage of a large DNA can be increased by simply terminating the reaction before completion; the result is called a partial digest. Fragment size can also

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