ChE 170L Laboratory
Exercise 4. Restriction Analysis of Plasmid DNA
Objectives
This experiment introduces the origin and the synthetic uses of restriction endonucleases,
enzymes that cleave double-stranded DNA. One of these applications is towards the
characterization of plasmid DNA according to the location of restriction sites. Students will
prepare agarose gels and apply the technique of gel electrophoresis to distinguish their plasmid
fragments based on size and corresponding electrophoretic mobility. A restriction map of the
plasmid DNA will be generated.
PreLab Questions 2
Please limit your answers to a few sentences IN YOUR LAB NOTEBOOK
1. If restriction enzymes are present in cells, why is their own DNA not cleaved as a result?
2. Many restriction enzymes cleave at unique sequences of 4-8 DNA base pairs that are
palindromic. What does this mean? What is the difference between “sticky ends” and
“blunt ends?”
3. Which type of restriction endonucleases (I, II, or III) are used for in vitro DNA
modifications? What enzyme is used to reconnect the bonds broken by these restriction
endonucleases?
4. Of the following, which is so temperature sensitive that it must be returned to ice or the
freezer after no more than a few seconds?
a. Ethidium bromide
b. Your plasmid solution
c. Molecular weight markers
d. Restriction enzymes
e. 6X loading buffer
f. restriction enzyme buffers (i.e. buffers 2 and 3)
5. What is the purpose of using the 6X running buffer when loading your samples on the
electrophoresis gel? How will increasing the agarose percentage affect the velocity of
DNA through the gel? Why?
Background
Bacteriophages, or “phages,” are viruses that infect certain species of bacteria. Phages
infect cells through insertion of viral DNA into the host organism. This DNA can be rendered
harmless to the cell through a unique system of restriction-modification. Cells distinguish their
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own DNA via methylation of various nucleotides at specific regions within the bacterial
chromosome. These regions consist of a unique sequence of DNA base pairs, ranging usually
from four to six bases in length, which are recognized by one of the cell’s methylase enzymes.
The enzyme covalently attaches a methyl group to one or more of the nucleotides within the
sequence, and thereby modifies the DNA. This methyl group acts as a marker to prevent a
corresponding restriction endonuclease enzyme from cleaving the strand.
If foreign,
unmethylated DNA enters the cell, the restriction endonuclease will recognize the unmodified
sequence and begin to degrade the DNA by cleaving the phosphodiester bonds of the double
stranded DNA backbone. Eventually, less specific exonucleases will fully break down the
invader. This remarkable bacterial defense system illustrates the extreme specificity of enzymesubstrate interactions. If an error in base pair recognition is made by either the methylase or the
endonuclease clipping the cell’s chromosomal DNA, the consequences can be fatal.
The ability of these enzymes to recognize and cleave distinct regions of DNA has been
exploited by biochemists and molecular biologists, and restriction enzymes are likely the single
most important tools in genetic engineering. Three types of restriction enzymes have been
isolated from bacteria. Types I and III contain both the methylase and endonuclease activity on
one enzyme. Type II enzymes contain only endonuclease activity, and these are the type used
for in vitro DNA modification. Hundreds of these enzymes have been isolated from
microorganisms, each recognizing a distinct region of DNA. Enzymes are classified based on
the genus, species, and strain of their native bacterium. Enzymes are numbered based on the
order of discovery. The first letter of the genus and first two letters of the species are italicized,
followed by the non-italicized letter of the strain, if relevant. For example, BamHI is an enzyme
native to Bacillus amyloliquefaciens H.
The recognition sequence of each isolated restriction enzyme has been deduced. For
most of these, the sequence is a palindrome. A unique feature of many of these is their ability to
produce cohesive, sticky ends, upon cleavage of the DNA. In this case, cleavage of the strands is
staggered about the center of symmetry of the recognition sequence. The resulting fragments
possess base pair complementarity. For example, SalI (isolated from Streptomyces albus)
recognizes the single-strand sequence: GTCGAC. But in double stranded DNA, this strand is
complementary to: CAGCTG. Arrows indicate the point of cleavage, so the enzyme’s action
is:
5’ …GT-C-G-A-C… 3’
3’ …C-A-G-C-TG… 5’
5’ …G
3’ …C-A-G-C-T
T-C-G-A-C… 3’
G… 5’
If this enzyme is allowed to digest a desired cloning vector, such as a plasmid, the plasmid will
open up and yield these sticky ends. Next, a gene that one wishes to insert into the plasmid can
be digested with the same enzyme to produce identical sticky ends. The plasmid and gene are
then combined in solution with DNA ligase, an enzyme that reconnects the bonds broken by the
restriction enzyme. A cloning vector containing the gene of interest results, and this can be
inserted into a host cell. Many plasmids have been developed as cloning vectors such that there
is only one restriction site for each of several different restriction enzymes. These cloning sites
provide only one location on the plasmid for insertion of the recombinant gene using a particluar
restriction enyzme. Other useful restriction enzymes produce blunt ends at their recognition site,
i.e. the DNA is cleaved at the center of symmetry of the sequence and the resulting fragments
cannot base pair. This would occur for SalI, shown above, if the enzyme cleaved between the
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central C-G bond on each strand. Different enzymes that recognize the same DNA sequence are
called isoschizomers.
The recognition sequence and cleavage site for each of the restriction enzymes used in
the experiment are given below (R = A or G, and Y = C or T). DNA sequences expressed this
way are always written in the 5 prime to 3 prime direction. This directionality describes the
manner in which the phosphodiester bonds of the DNA backbone are chemically linked.
Eco RI
Ssp I
Pvu I
Afl III

G-A-A-T-T-C
C-T-T-A-A-G


A-A-T-A-T-T
T-T-A-T-A-A


C-G-A-T-C-G
G-C-T-A-G-C


A-C-R-Y-G-T
T-G-Y-R-C-A

Some plasmids possess more than one restriction site per enzyme, in which case multiple
DNA fragments will be produced upon digestion. If two restriction enzymes are allowed to
digest the plasmid, a so-called double digest, information can be obtained regarding the relative
proximity of each enzyme’s restriction site(s). Single and double digests can be performed using
multiple enzymes, allowing one to generate a restriction map of a given plasmid. A restriction
map of the plasmid shows the distances between the restriction sites of all enzymes used in the
experiment. Recall from the previous Exercise, that the electrophoretic mobility of a DNA
fragment though an agarose gel is a function of the fragment size. Hence, gel electrophoresis can
be used to determine the sizes of the DNA fragments that result from the digestion of plasmid
DNA with one or more restriction enzymes.
PROCEDURE
NOTE: Restriction enzymes are extraordinarily expensive and must be handled with great care.
Do not contaminate any of the samples, and do not use pipette tips used with enzymes more than
once. Enzymes are generally stored at –20 °C. During this procedure, the enzymes should not
be held out of the ice for longer than a few seconds.
Restriction Digests
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ChE 170L Laboratory
1. Prepare a chart detailing the quantity of each solution to be added to each reaction, and in
what order. This chart will prevent much confusion. This should be done prior to the lab,
since it takes several minutes to prepare, and would waste laboratory time.
2. Remove the microcentrifuge tube containing the plasmids you isolated in the previous lab
from the refrigerator.
3. Obtain 9 - 1.5 ml microcentrifuge tubes and number them 1-9. These tubes correspond to the
digests you will be performing. Please note that different buffers need to be used with
different restriction enzymes.
1.
2.
3.
4.
5.
6.
7.
8.
9.
A
B
C
D
A+B
ABD
C+B
C+D
D+A
Enzymes
Buffer
Eco RI
Ssp I
Pvu I
Afl III
Eco RI + Ssp I
Eco RI + Ssp I + Afl III
Pvu I + Ssp I
Pvu I + Afl III
Afl III + Eco RI
3
2
3
3
2
2
2
3
3
4. In each 1.5-ml microcentrifuge tube, combine the following in order:
Enough water to make 20 l total digest volume
4 l of your pUC-19 DNA prep from Exp. 3
2 l of the appropriate 10X buffer
1 l of enzyme for single digests, 1 l of each enzyme for multiple digests
Plan how you will be changing pipette tips. Water and then DNA can be added to all of your
tubes with the same tip. Tips should be changed before each time you pull from a communal
buffer or enzyme tube (even if you're still using the same buffer).
Restriction enzymes are extremely temperature sensitive and should be out of the ice for no
more than a few seconds at a time. Since the enzymes and buffers are shared, everyone
depends on the courtesy of everyone else.
Correct Pipetman usage is absolutely vital for this lab to work. Surface tension forces
dominate with the tiny volumes are to be pipetted. Make sure the volumes you pull up are
consistent. Do this by monitoring the liquid level in the pipette tip. Make sure you deliver
the volume directly to the bottom of the microtube, and see all the liquid come out of the tip.
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ChE 170L Laboratory
Sterile technique is not being used, and the buffers are all relatively harmless, so look closely
at what you're doing.
5. Incubate the reaction mixtures for 1 hour at 37 °C. Remove samples from the incubator and
prepare sample for DNA gel.
Preparing, Running, and Viewing a 1% Agarose Gel
6. Prepare a 1% agarose gel as described in Exercise 3. While the gel is cooling, prepare your
samples as described below.
7. Prior to the lab period, prepare a table which lists the lane number, name, and components
of each sample that will be run on the gel. Each of your digests will be run in a separate lane.
For the undigested sample, add water instead of buffer and enzymes. Use 4 l of uncut
plasmid. You should have a total volume of 20 l for each sample, not including the loading
buffer. Use water to obtain this volume for the uncut sample and the standards. You will run
2 lanes of molecular weight standards (5 l) on the gel. Standards do not require addition of
loading buffer. You will need to add 4 l 6X loading buffer to each 20 l digest and uncut
plasmid.
8. Remove your digestions from the incubator and briefly centrifuge them so that any water
which may have condensed on the sides and top of the tube will be collected at the bottom.
9. Obtain three 1.5 ml centrifuge tubes to prepare your uncut sample and the molecular weight
standards. Arrange all of the tubes on your microcentrifuge tube rack in the order in which
you have decided to load them so that you will not become confused later.
10. Add 4 l 6X loading buffer to each of your digests and uncut plasmid. You will load 15 l
of this into each lane.
11. Prepare your samples according to the table you prepared in Step #7.
12. Once the gel has cooled, assemble the electrophoresis apparatus and load the gel as described
and demonstrated in the previous Exercise.
13. Run the gel for 1 to 1.5 hours at about 100 volts. Stain, view, and photograph the gel as
described and demonstrated in the previous Exercise.
Guidelines for Analysis & Conclusions Section
(Remember, these are points you should consider and include in your analysis. This section,
however, need not be limited to these specific guidelines.)
1. The final objective of this lab, and arguably of labs 2-4 combined, is the preparation of a
restriction map for the plasmid pUC19. Prepare a standard plot using the molecular weight
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ChE 170L Laboratory
marker standards. (Refer to Question #5 in the “Guidelines…” section of Exercise 3 if
you’ve forgotten how to do this.) The molecular weight standard is the same as the one you
used previously. Estimate the sizes of your plasmid fragments from each of the digests.
After you have determined the size of each band, the restriction mapping becomes a
relatively simple exercise in logic. Prepare a restriction map of your plasmid.
2. Two double-stranded DNA molecules, 1 and 2, are cleaved by the restriction endonuclease
EcoRI. (One strand of each DNA fragment is shown below. Use the base-pairing rules to
determine the sequence of the complementary strand.) The resulting fragments are mixed,
allowed to recombine, and covalently closed with DNA ligase. List all of the possible
reaction products, assuming that none of the base pairs dissociate. Assume that each product
will have only one copy of each fragment.
DNA 1: (5’) CGATAGAATTCAGTCAA (3’)
DNA 2: (5’) GAATTCGGTTCGAATTCG (3’)
3. A new strain of bacteria was recently found in the steam vents of U.C. Berkeley. Hopes are
high that a plasmid isolated from this remarkably resilient organism carries genes conferring
resistance to antibiotics. You decide to generate a restriction map in order to characterize the
plasmid. The sizes of the restriction fragments were determined from their electrophoretic
mobilities on agarose gels. From the data shown below, construct the plasmid’s restriction
map.
Restriction Enzymes
Bam HI
Bst BI
Afl III
Alw NI
Bam HI + Bst BI
Bam HI + Afl III
Bam HI + Alw NI
Bst BI + Afl III
Bst BI + Alw NI
Afl III + Alw NI
Fragment Sizes (kb)
7627
7627
5583, 2044
4406, 1741, 1480
4865, 2762
3431, 2152, 2044
3847, 1741, 1480, 559
4914, 2044, 669
3321, 1741, 1480, 1085
3990, 1593, 1480, 416,
148
4. What is the equation for the probability p of finding a specific sequence of n bases in a
random fragment of DNA? Use your equation to predict the number of EcoRI restriction sites
in a plasmid of length 4.1 kb.
5. SV40 DNA is a circular molecule of 5243 bp that is 40% G+C. In the absence of sequence
information, how many restriction cuts would EcoRI, Ssp I, Afl III, and Pvu I be expected to
make in SV40 DNA? Show your work.
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6. Recalling what was learned about supercoiling in the previous experiment, explain the
band(s) obtained from subjecting the whole, undigested plasmid to gel electrophoresis.
7. Discuss the chemistry of ethidium bromide binding to DNA. This should give you insight
into the carcinogenic properties of EtBr.
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ChE 170L Laboratory
EQUIPMENT AND REAGENTS
Electrophoresis unit. Includes power supply, box, plate, and sample comb.
1.5ml microcentrifuge tubes
Sterile micropipet tips
Gel camera
Polaroid Film for gel pictures
UV light box
Restriction Enzymes from New England Biolabs (Commercial Preps @ 5000-20,000 units/ml
sold in 500 to 20,000 unit aliquots): (a) EcoRI, (b) Ssp I, (c) AlwN I (d) BglI. 10X
buffers supplied with enzymes.
Commercial Molecular Weight Marker, 1 kb DNA ladder from Life Technologies
Ethidium Bromide (EtBr): Stock Solution at 10 mg/ml
Agarose (Electrophoresis-grade)
10X loading buffer (see Ex 3 for composition)
50X TAE Gel Electrophoresis Buffer (see Ex 3 for composition)
REFERENCES
Bailey, J.E., and Ollis, D.F. (1986) Biochemical Engineering Fundamentals, 2nd Ed., pp. 340344, McGraw Hill, Inc., New York.
Blanch, H.W., and Clark, D.S. (1996) Biochemical Engineering, pp. 533-538, Marcel Dekker,
Inc., New York.
Voet, D., Voet, J.G. (1990) Biochemistry, pp. 824-829, John Wiley and Sons, New York.
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