Y

Polylinker lacZ+

pUC19

ampR

ampR

18.7 The pUC19 plasmid is a typical cloning vector. It contains a cluster of unique restriction sites, an origin of replication, and two selectable markers—an ampicillin-resistance gene and a lacZ gene.

ward method cannot be used. Second, this technique often leads to undesirable products. The sticky ends of the plas-mid are complementary to each other; so the two ends of the plasmid will often simply reanneal, reproducing the intact plasmid. Alternatively, the two complementary ends of the cleaved foreign DNA may anneal; or several pieces of foreign DNA or several plasmids may join. However, these undesirable products do not constitute a serious problem if an efficient method is used for screening bacterial cells for the presence of a recombinant plasmid.

Another method for inserting DNA into a plasmid (a method that gets around the problem of undesired products) is tailing (IFigure 18.8b). In this procedure, comple mentary sticky ends are created on blunt-ended pieces of DNA. The plasmid and the foreign DNA are first cut by any restriction enzyme. If the restriction enzyme produces sticky ends, these ends are removed by an enzyme that digests single-stranded DNA. Alternatively, the plasmid and foreign DNA can be cut by a restriction enzyme that produces blunt ends.

Once the plasmid and the foreign DNA have blunt ends, single-stranded sticky ends are added by an enzyme called terminal transferase, which adds any available nucleotides to the 3' end of DNA in a template-independent reaction. For example, terminal transferase and deoxyadenosine triphosphate (dATP) might be mixed with the plasmid DNA, creating poly(A) single-stranded tails on the 3' ends of the plasmid. Terminal transferase and deoxythymidine triphosphate (dTTP) could be mixed with the blunt-ended foreign DNA fragments, creating poly(T) single-stranded tails on their 3' ends. The poly(A) tail of the plasmid would be complementary to the poly(T) tail of the foreign DNA, allowing them to anneal and connecting the plasmid and foreign DNA together. DNA polymerase can be used to fill in any missing nu-cleotides, and DNA ligase can be used to seal the nicks in the sugar - phosphate backbone.

One advantage of tailing is that it prevents the production of the undesired products created by restriction cloning: the single-stranded ends of the plasmid are complementary only to the single-stranded ends of the foreign DNA. Another advantage is that identical restriction sites are not required in plasmid and foreign DNA; any restriction site can be used for cleavage. But tailing has several disadvantages of its own. First, it destroys the restriction site used to cut the original molecule, preventing later cleavage by the same restriction enzyme to retrieve the foreign DNA. Second, the new nucleotides (the complementary tails) introduced at the junctions between plasmid and foreign DNA sometimes interfere with the function of the cloned DNA.

A third method of inserting fragments into plasmids is to use the enzyme T4 ligase, which is capable of connecting any two pieces of blunt-ended DNA. Like tailing, this

(a) Restriction cloning

(b) Cloning by tailing

(c) Cloning by using linkers

Plasmid

Eco RI

Eco RI

Foreign DNA

0 0

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