USING RESTRICTION ENZYMES
The DNA sequences used in the construction of recombinant DNA molecules can originate from
species. For example, plant DNA may be joined to bacterial DNA, or human DNA may be
fungal DNA. In addition, DNA sequences that do not occur anywhere in nature may be
created by the
chemical synthesis of DNA, and incorporated into recombinant molecules. Using
recombinant DNA technology and synthetic DNA, literally any DNA sequence may be
introduced into any of a very wide range of living organisms.
Gene cloning and genetic engineering were made possible by the discovery of enzymes that cut
DNA molecules at a limited number of specific locations. These enzymes, called restrict
enzymes, were discovered in bacteria in the late 1960s. In nature, these enzymes protect the bacteria
against intruding DNA from other organisms, such as viruses or other bacterial cells. They work by
cutting up the foreign DNA, a process called rest
riction. Most restriction enzymes are very specific,
recognizing short nucleotide sequences in DNA molecules and cutting at specific points within these
sequences. The bacterial cell protects its own DNA from restric
tion by adding methyl groups
CH3) to adenines or cytosines within the sequences recognized by restriction enzymes. Hundreds
of different restriction enzymes have been identified and isolated.
Most restriction sites are symmetrical. The same sequence of four
to eight nucleotides
is found on both strands but running in
opposite directions. Restriction enzymes cut the bonds of both
strands, often in a staggered way. Since the target sequence usually
occurs many times in a long DNA molecule, an enzyme will make
many cuts. Copies o
f a DNA molecule always yield the same set of
restriction fragments when exposed to that enzyme. In other words,
a restriction enzyme cuts a DNA molecule in a reproducible way.
Notice that the restriction fragments are double
h at least one single
stranded end, called a sticky end.
These short extensions will form hydrogen
bonded base pairs with
stranded stretches on other DNA molecules
cut with th
e same enzyme. The unions formed
in this way are only
orary, because only a few hydrogen bonds hold the fragments
together. The DNA fusions can be made permanent, however, by
the enzyme DNA ligase, which seals the strands by forming
MAKING RECOMBINANT DNA VECTORS
With a restriction enzyme
and DNA ligas
e, we can make a
. The original
is called a cloning vector, defined as a DNA molecule that can carry foreign DNA into a cell
and replicate there. Bacterial plasmids are widely use
as cloning vectors. Recombinant
produced by splicing restriction fragments from foreign DNA into plasmids isolated from bacteria
can be returned relatively easily to bacteria. Then, as a bacterium carrying a recombinant plasmid
reproduces, the plasmid
replicated within it.
The resulting cell clone, which appears as a colony,
contains multiple copies of the foreign DNA.
1. Why do bacteria have restriction
1a. Why don’t the restriction enzymes cut the
bacteria’s own DNA?
nk of a symmetrical pattern that a restriction enzyme
might use as a cut site. It must read the same going forward on
the top strand and backwards on the bottom strand.
3. Most restriction enzymes cut in a way that is different than
we learned in cl
ass. How do most restriction enzymes cut?
3a. Why is this cut pattern important?
4. Which enzyme is used to glue the DNA pieces together?
5. Why are both the plasmid and the gene of interest cut using
the same restriction enzyme?
6. What are some recombinations that could easily happen to
result in a plasmid being created that does NOT contain your
desired result? Think of at least 2.
7. When would PCR be used in the process of creating a
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