Inserting Genes into Vectors

The simplest way to insert a segment of DNA into a vector is to cut both the target DNA and the vector with the same restriction enzyme. If a restriction enzyme that generates sticky ends is used, the vector and the insert will have matching overhangs. A mixture of the two is treated with DNA ligase, which links together DNA strands. The result is the ligation of the target DNA fragment into the vector as shown in Figure 22.11. If a restriction enzyme that generates blunt ends is used, ligation is more difficult and T4 ligase must be used as discussed above.

This procedure relies on the vector possessing only one site for the chosen restriction enzyme. If there were more than one cut site in the vector the restriction enzyme amp gene Gene conveying resistance to ampicillin and related antibiotics and encoding beta-lactamase. Same as bla gene ampicillin A widely used antibiotic of the penicillin family beta-lactamase (b-lactamase) Enzyme that degrades beta-lactam antibiotics, including penicillins and cephalosporins bla gene Gene conveying resistance to ampicillin and related antibiotics and encoding beta-lactamase. Same as amp gene

ColEI plasmid Small multicopy plasmid of Escherichia coli that forms the basis of many cloning vectors widely used in molecular biology

FIGURE 22.11 Insertion of DNA into Cloning Vector

To insert the gene of interest into a plasmid vector, both the vector and the gene of interest should have compatible sticky ends. To achieve this both the gene and the vector must be digested with the same restriction enzyme. The two pieces are mixed together with DNA ligase, which joins the ends yielding a closed double-stranded circular plasmid carrying the gene of interest.

Vector

Gene to be cloned

Cut vector open

Restriction

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