Restriction Enzyme Digest and Gel
Electrophoresis Field Trip
Background Information
Restriction enzymes are proteins that cut double stranded DNA at
specific recognition sites. They were first proposed in the early
1960s, W. Arber and D. Dussoix to explain the way bacteriophage
(viruses that infect bacteria) could infect some strains of bacteria
but not others. Restriction enzymes are isolated from bacteria.
The bacteria use them as protection against the invasion of foreign
DNA. In 1968, W. Arber and S. Linn and then M. Meselson and R. Yuan
purified similar enzymes that were able to cut DNA, but these early
enzymes cleaved the DNA at random positions. In 1970, H.O. Smith,
K. W. Wilcox and T. J. Kelly purified and characterized the recognition
and cleavage site of a more useful enzyme, Hind II, an enzyme that
cut at a specific recognition site every time. This enzyme was used
by Daniel Nathans to cut the circular genome of Simian Virus 40
(SV40) to generate the first restriction map. The ability to cut
DNA at specific sequences became the first step toward molecular
cloning.
In the Restriction Enzyme Digest and Gel Electrophoresis of DNA
field trip, the students cut lambda DNA with 4 different restriction
enzymes and use gel electrophoresis to visualize the DNA. Molecular
biology laboratory skills and equipment, as well as laboratory safety,
will be discussed and used in this lab.
Lambda DNA is used in this experiment. Lambda is a bacteriophage.
The isolated DNA is linear, 48,502 base pairs long, and has recognition
sites for many different restriction enzymes. It is commonly used
for molecular weight, size markers in gel analysis of DNA, as well
a substrate in restriction enzyme activity assays.
In this lab, we cut lambda DNA with four different restriction
enzymes in the appropriate buffers. This is a restriction digest.
Restriction enzymes act like scissors to cut DNA into pieces. Different
restriction enzymes (and there are hundreds) recognize and cut different
DNA sequences. When the DNA is cut, the sizes of the DNA fragments
correspond to the distances (in base pairs) between restriction
sites. The reaction is incubated at the enzyme's optimum temperature
to digest the DNA. This usually takes about 45 minutes. During this
time agarose gels will be prepared.
Purified agarose is a powder that is insoluble in water (or buffer)
at room temperature but dissolves in boiling water (or buffer).
As it cools, agarose undergoes polymerization. The sugar polymers
crosslink with each other and cause the solution to "gel",
much like JELLO. Higher concentrations of agarose give firmer gels.
If you were inside a gel, it would resemble a very dense spider
web. If you were a small fragment you could easily crawl through
the spaces between the webs (they are too tough for you to just
pull out of the way) but as you increase in length, it gets harder
and harder for you to fit through the spaces. We use a 0.7% agarose
gel in 0.5x TBE buffer (Tris/Borate/EDTA) for this experiment. The
agarose is kept molten at 55° C until the gels are poured. Gels
are prepared by pouring the molten agarose into casting trays around
a six-well comb and allowing them to solidify. The comb is removed
from the solidified gel and the DNA is loaded into the wells left
by the comb.
The gel box is prepared by pouring 0.5x TBE into the chambers and
placing the gel in the box. In order to visualize the DNA in the
gel, a chemical called Ethidium Bromide (EtBr) is added to the gel. EtBr intercalates into the DNA and fluoresces
when it is exposed to ultraviolet light. EtBr is a mutagen and must
be handled carefully. We always wear gloves, lab coat and safety
glasses when using EtBr
The digested DNA and some DNA markers will be mixed with a loading
dye containing Tris-HCl, EDTA, 10% Ficoll, and 3 dyes. The samples
are loaded into the agarose gel wells and then electrophoresed by
connecting the gel box to a power supply. Electrophoresis is a laboratory
technique used the to separate charged molecules. DNA is negatively
charged and moves, under the force of an electric current, through
the matrix of the agarose. Molecules separate by size, with the
smaller ones moving more rapidly through the gel than the large
ones.
After running the gel, the DNA is visualized using medium wavelength
ultraviolet (UV) light (we always wear safety glasses while using
a UV light source). The EtBr binds to the DNA during electrophoresis.
When the gel is exposed to UV light, the DNA bands fluoresce in
a distinctive pattern of DNA fragments, one band for each different
size fragment. We take a photograph of the gel so that it is easier
to study the banding pattern.
If you have any questions or would like more information before
you bring your students to the BTCI for this field trip, please
give us a call. Alternatively, bring your questions along and we
can discuss them during the lab. We look forward to seeing you and
your group on your scheduled field trip day. Thank you for your
interest in BTCI's Biotechnology Field Trips program.
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