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CR Restriction Enzymes
CR Restriction Enzymes
Introduction
Recombinant DNA technology or “gene splicing,” as it is frequently called, often involves
insertion of selected DNA sequences from a variety of sources into plasmids which are then
transformed into bacteria. The resultant recombinant bacterial strain may now express the
plasmid genes as well as whatever foreign gene(s) that was inserted.
This process would not be possible without accurate and predictable ways of cutting the DNA of
the plasmid and the foreign source. Restriction endonuclease enzymes provide this service.
Restriction endonucleases, often called restriction enzymes, cut DNA within the molecule by
hydrolyzing the phosphodiester bonds between the nucleotides. Restriction enzymes were
discovered in bacteria and there are now more than 1200 known restriction enzyme types. These
enzymes are named using a simple system. EcoRI, for example, was isolated from E. coli and
was the first enzyme isolated from a particular strain, hence the designation of I. HaeIII was
isolated from Haemophilus aegyptius. HindIII was isolated from Haemophilus influenzae, and
was the third enzyme discovered in a particular strain.
Usually, restriction enzymes only cut the DNA at or near a very specific nucleotide sequence
known as a recognition site. This “restrictive” nature of these enzymes allows molecular
biologists to pin-point exactly where the DNA is to be cut. In many cases, the recognition site of
a restriction enzyme consists of a palindromic sequence where the order of the bases is read the
same on each side of the helix. The two sides of the helix are antiparallel and the recognition
sequence reads from 5’ to 3’ on either side of the helix. For example the recognition site for
EcoRI is as follows;
GAATTC
CTTAAG
Some restriction enzymes cut both sides of the helix at the same point. HaeIII is such an enzyme
and the fragments produced are “blunt” ended. Many restriction enzymes make asymetric cuts
in the helix. EcoRI cuts DNA at the recognition site in the following fashion;
G↓AATTC
CTTAA↑G.
This type of asymetric cut leaves a single stranded sequence hanging on either 5’ or 3’ end.
1. When the incubation of the pUC19 digest ends, transfer this tube to ice for 5 minutes.
2. Add 2μl of loading dye- Label this tube pUC19/EcoRI. Place this tube on ice.
3. When the incubation of the pRAS2 digest ends, add 2μl of loading dye directly to this digest-
flick the tube to mix and microfuge for a few seconds. Transfer this tube to ice.
4. When the incubation of the genomic Wheat Germ DNA digest ends, transfer this tube to ice
for 5 minutes.
5. Add 2μl of loading dye. Flick this tube and briefly spin and label it Genomic/EcoRI. Place this
tube on ice.
6. When the incubation of the Lambda DNA digest ends, transfer this tube to ice for 5 minutes.
7. Add 2μl of loading dye. Label this tube Lambda/EcoRI and place on ice.
Note: At this point each group should have 4 tubes (4 digests) which contain loading dye. These
tubes can be stored on ice overnight.
1. Prepare a 1% Agarose Gel (eight well comb) and allow it to stand for 20 minutes
2. After removing the dams, add the chamber buffer. Then, after a few minutes to allow the gel
to soften, remove the comb by pulling straight up. (Note- The instructor can save time by
doing steps 1 and 2 prior to class),
3. Load 20μl of each sample (the one kilobase ladder and 4 digests) into the wells in the order
indicated.
4. After the samples have been loaded, close the lid, connect the power supply, and switch on.
Set the voltage at 110 volts.
5. Electrophorese for about 30 minutes until the dye has reached midway between the second and
third red stripes. Switch off the power supply and open the lid.
6. Wearing gloves, remove the gel and stain with methylene blue in a tray for 30 minutes and
move the gel to a tray of distilled water for 15 minutes to destain.
7. After staining, place the gel in a tray (half of an empty tip box ) using a spatula and bring to
the light box.
8. The instructor will photograph your gel. (You may destain overnight if needed.)
9. After photographing, discard the gel and gloves in the waste container for proper disposal.
Discussion Questions
1. How many places does EcoRI cut pUC19? How many fragments are produced?
2. Approximate the size of the pUC19 fragment(s) using the kilobase ladder. Your instructor will
show you a graphing method using semi-log paper.
3. How many places does EcoRI cut pRAS2? How many fragments are produced? Approximate
the sizes of these fragments.
4. pRAS2 is a modification of pUC19 in which a segment of yeast DNA is added. Do your
results support this? Explain?
5. The digest of Lambda DNA by EcoRI produces 6 fragments of the following sizes; 21226 bp,
7421 bp, 5804 bp, 5643 bp. 4878 bp, 3530 bp. The 5804 and 5643 fragments may appear as a
single heavy band. Why? Using the graphing technique and the one kilobase ladder bands and
their migration distances, approximate the sizes of the lambda fragments and compare these to
the actual sizes listed above. Determine a percent error for each fragment measurement.
6. How many fragments are produced by digestion of your genomic Wheat Germ DNA
byEcoRI? Is this what you expected? Explain.
1. This exercise requires one double period and a single period the next day (or within a few
days). Each student group should contain four students. One student member of each group
should work on one digest and follow all the procedures for that digest up to and including
preparation for electrophoresis. (There are four digests- pUC19, pRAS2, lambda phage, and
genomic (if you isolated this in a previous lab). All digests should be prepared, incubated,
prepared for electrophoresis and stored the first day.
2. The gels should be made the second day and may be poured ahead of time and stored under
chamber buffer.
3. The gels may not be finished running during the class period of the second day, so the teacher
may have to stain the gels and photograph them. The gels should be stained and
photographed right after being run since the bands become diffuse with time.
4. As always, make certain that the students are familiar with all transfer techniques,
electrophoresis procedures and care and cleaning of equipment. Stress safety when dealing
with high voltage.
Materials List for Restriction Enzyme Exercise (for each group of four students)