Enzyme

Cut ends of insert

Dna ligase

Ligate gene and vector together

Ligate gene and vector together

would cut it into multiple fragments. Furthermore, we must avoid inserting the cloned gene into any of the genes needed by the plasmid for its own replication and survival within the cell. Moreover, since there are many different restriction enzymes, it would be convenient to have a wide range of restriction recognition sites in the vector. These issues are all resolved by inserting a polylinker, or multiple cloning site (MCS), into the cloning vector.This is a stretch of artificially synthesized DNA, about 50 base pairs long, which contains cut sites for seven or eight widely used restriction enzymes. This not only allows a wide choice of restriction enzymes, but ensures that the insert does not damage the plasmid and goes into more or less the same location each time (Fig. 22.12).

The remainder of the plasmid should not contain any cut sites for any of the enzymes represented in the polylinker. One way to ensure this is to choose only enzymes with zero cut sites in the original plasmid. Alternatively, we can get rid of unwanted cut sites by the approach shown in Figure 22.13. Due to spontaneous mutation (see Ch. 13 for mutations), occasional plasmids will suffer a base change within the cut site that needs to be eliminated. This will abolish recognition of the site by the restriction enzyme. The question is how to find this one rare mutant plasmid. First plasmid DNA is prepared and treated with the restriction enzyme in question. The plasmid DNA is then transformed into fresh bacterial cells without re-ligating the break. Wild type bacteria rapidly degrade incoming linear DNA; therefore, the majority of plasmids will be destroyed by this procedure. Those few that have lost the cut site by mutation will remain circular and survive.

miilliple cloning site (MCS) A stretch of artificially synthesized DNA that contains cut sites for seven or eight widely used restriction enzymes. Same as polylinker polylinker A stretch of artificially synthesized DNA that contains cut sites for seven or eight widely used restriction enzymes. Same as multiple cloning site (MCS)

FIGURE 22.12 Polylinker or Multiple Cloning Site

Many plasmid vectors contain an artificial region of DNA that has many different restriction enzyme sites. Such polylinkers or multiple cloning site are designed so that all the restriction enzyme sites in the polylinker are unique, and the corresponding enzymes only cut the plasmid once.

Recognition site for restriction enzyme 1

Recognition site for restriction enzyme 3

Recognition site for restriction enzyme 5

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