Steps for Using Bacteria and Plasmids to Clone Genes

Recombinant DNA molecules are only useful if they can be made to replicate and produce a large number of copies. A typical gene-cloning procedure includes the following steps (See Campbell, Figure 19.3):

Step 1: Isolation of two kinds of DNA.

• Bacterial plasmids and foreign DNA containing the gene of interest are isolated.
• In this example, the foreign DNA is human, and the plasmid is from E. coli and has two genes:

  --> amp^R that confers antibiotic resistance to ampicillin.

  --> lacZ that codes for beta-galactosidase, the enzyme that catalyzes the hydrolysis of lactose

• Note that the recognition sequence for the restriction enzyme used in this example is within the lacZ gene.

Step 2: Treatment of plasmid and foreign DNA with the same restriction enzyme.

• The restriction enzyme cuts plasmid DNA at the restriction site, disrupting the lacZ gene.
• The foreign DNA is cut into thousands of fragments by the same restriction enzyme; one of the fragments contains the gene of interest.
• When the restriction enzyme cuts, it produces sticky ends on both the foreign DNA fragments and the plasmid.

Step 3: Mixture of foreign DNA with chopped plasmids.

• Sticky ends of the plasmid will base pair with complementary sticky ends of foreign DNA fragments.

Step 4: Addition of DNA ligase.

• DNA ligase catalyzes the formation of covalent bonds, joining the two DNA molecules and forming a new plasmid with recombinant DNA.

Step 5: Introduction of recombinant plasmid into bacterial cells.

• the naked DNA is added to a bacterial culture.
• Some bacteria will take up the plasmid DNA by transformation.

Step 6: Production of multiple gene copies by gene cloning and selection process for transformed cells.

• Bacteria with the recombinant plasmid are allowed to reproduce, cloning the inserted gene in the process.
• Recombinant plasmids can be identified by the fact that they are ampicillin resistant and will grow in the presence of ampicillin.

Step 7: Final screening for transformed cells.

• X-gal, a modified sugar added to the culture medium, turns blue when hydrolyzed by beta-galactosidase. It is used as an indicator that cells have been transformed by plasmids containing the foreign insert.
• Since the foreign DNA insert disrupts the lacZ gene, bacterial colonies that have successfully acquired the foreign DNA fragment will be white. Those bacterial colonies lacking the DNA insert will have a complete lacZ gene that produces beta-galactosidase and will turn blue in the presence of X-gal.

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