Steps for Using Bacteria and Plasmids to Clone
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,
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:
- --> ampR that confers antibiotic resistance
- --> 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
- 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
- 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
Step 5: Introduction of recombinant plasmid into bacterial
- the naked DNA is added to a bacterial culture.
- Some bacteria will take up the plasmid DNA by
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
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.
From Campbell's Student Study Guide