2005 by The International Union of Biochemistry and Molecular Biology

Printed in U.S.A.

BIOCHEMISTRY

MOLECULAR BIOLOGY EDUCATION

Vol. 33, No. 1, pp. 28 33, 2005

AND

Laboratory Exercises
Quantification of DNA by Agarose Gel Electrophoresis and Analysis
of the Topoisomers of Plasmid and M13 DNA Following Treatment
with a Restriction Endonuclease or DNA Topoisomerase I
Received for publication, June 8, 2004, and in revised form, July 20, 2004
John W. Tweedie and Kathryn M. Stowell
From the Institute of Molecular Biosciences, Massey University, Palmerston North, New Zealand

A two-session laboratory exercise for advanced undergraduate students in biochemistry and molecular
biology is described. The first session introduces students to DNA quantification by ultraviolet absorbance
and agarose gel electrophoresis followed by ethidium bromide staining. The second session involves
treatment of various topological forms of DNA with a restriction endonuclease and with a DNA topoisomerase. This session introduces students to the concept of DNA topoisomers, to the properties of different
forms of DNA, and to the activity of restriction endonucleases and topoisomerases toward these forms. The
exercise also involves measuring the size of linear duplex fragments of DNA by comparison of mobility with
a ladder of double stranded DNA of known sizes.
Keywords: DNA topoisomers, restriction endonuclease, DNA gel electrophoresis, DNA topoisomerases, DNA
quantification.
the same DNA. This gives the students a feeling for the
amount of DNA that can be visualized by this procedure.
The experiment also introduces the concept of topoisomers because, in addition to covalently closed supercoiled
circular DNA, most plasmid preparations also contain substantial amounts of relaxed circles (generated by nicking of
one strand) and smaller amounts of linear DNA generated
by double-strand breaks. Students are faced with the situation where a preparation, supposedly containing a single DNA species, shows two or three distinct bands on
agarose gel electrophoresis.
In the second part of the practical, plasmid and M13
DNA are treated with the restriction endonuclease EcoRI
and eukaryotic DNA Topoisomerase I [3], and the reaction
products are examined by agarose gel electrophoresis.
Both covalently closed circular (RFI) and single-strand circular (SS!) M13 DNA samples are treated. The results
from this part of the practical allow the students to interpret and identify the multiple bands seen in gel electrophoresis of plasmid DNA in the first part of the experiment.
They can also interpret their results in terms of the effects of Topoisomerase I and EcoRI on double-stranded
and single-stranded DNA. The reading list for the practical includes material on the functional aspects of DNA
topology and its manipulation. This reinforces material
on DNA topology contained in the corresponding lecture
course.
The experimental component of the exercise can be
completed within two 3-h laboratory periods and is carried
out by groups of two or three students. The total number
of students accommodated is only limited by the availability of equipment for gel electrophoresis.

When dealing with DNA there are two fundamental concepts that students in molecular biology must have a
feeling for: the small quantities of DNA involved in most
manipulations and the topological properties of these molecules. These concepts are particularly relevant when examining circular and linear DNA molecules of plasmids and
bacteriophage by gel electrophoresis. Students must have
an appreciation of the relation between the amount of DNA
and its appearance after agarose gel electrophoresis and
staining with ethidium bromide. They also need to be
aware of the different mobilities exhibited by DNA molecules differing only in their topological form. In addition to
these practical aspects, the role of DNA topology and its
manipulation in DNA replication, transcription, and other
metabolic transactions of DNA is fundamental to an understanding of these topics.
We have designed a two-part student laboratory exercise to illustrate and reinforce these concepts and to provide an experimental background to lectures on DNA topology and topoisomerases in a third-year undergraduate
course in the Institute of Molecular BioSciences. The theoretical material is based on current biochemistry and
molecular biology texts [1, 2]. About 500 students have
used the exercise over the past 10 years.
The first part of the experiment involves the quantification of plasmid DNA by ultraviolet (UV)1 absorbance followed by agarose gel electrophoresis of serial dilutions of
To whom correspondence should be addressed: Institute of
Molecular Biosciences, Massey University, Palmerston North,
New Zealand. E-mail: j.tweedie@massey.ac.nz.
1
The abbreviations used are: UV, ultraviolet; TAE, Tris acetateEDTA; TE, Tris-EDTA; DTT, dithiothreitol.

28

This paper is available on line at http://www.bambed.org

29
OUTLINE OF THE PRACTICAL

First Practical Session

Each group of students is provided with a sample of a
plasmid with a single EcoRI site and which is about 3 kbp.
We have used pUC118 for the data shown here. They are
told the approximate concentration of the plasmid and the
A260 for double-stranded DNA. After calculating an appropriate dilution they prepare a 1-ml aliquot, which is used to
obtain A260 and A280 readings. They now calculate the
actual DNA concentration of their sample and make a
series of dilutions that will give known amounts of DNA per
10 !l of TE buffer from 1 !g down to 1 ng. Ten-microliter
aliquots of the dilutions are mixed with 2 !l of loading dye,
and the mixed samples were loaded into the wells of an
agarose gel. Following electrophoresis, the gels are
stained with ethidium bromide, visualized under UV light,
and the image recorded for return to the students for their
interpretation.

Second Practical Session

In this session, the students are provided with samples
of the plasmid used in the first session and samples of
double-stranded circular (RFI) and single-stranded circular
(SS!) DNA of the bacteriophage M13.
Each group of students treats the covalently closed
circular (ccc, RFI) and the single-stranded circular (SS!)
DNA of bacteriophage M13 with DNA Topoisomerase I
and with the restriction endonuclease EcoRI. Plasmid DNA
(pUC118) is also treated with EcoRI and with DNA Topoisomerase I. Following digestion, the various forms of M13
and pUC118 DNA are separated by agarose gel electrophoresis in the absence of ethidium bromide. The gels are
stained following electrophoresis and visualized and recorded as for the first session.
MATERIALS AND METHODS

Buffers & SolutionsTris acetate-EDTA (TAE) and Tris-EDTA

(TE) buffers (10") are prepared as described by Sambrook and
Russell [4]. Topoisomerase 1 buffer (10") contains Tris-HCl (0.5
M), pH 7.5; KCl (0.5 M); MgCl2 (0.1 M); EDTA (1.0 mM); dithiothreitol
(DTT) (5.0 mM), and bovine serum albumin (300 !g/ml). Topoisomerase storage buffer (1") contains potassium phosphate (30
mM, pH 7.0); DTT (5.0 mM); EDTA (0.1 mM); glycerol (50% v/v); and
Triton X-100 (0.1% w/v). React 3 buffer was obtained from
Invitrogen (Carlsbad, CA). DNA gel sample solution contains
0.1% (w/v) bromphenol blue; 10% (v/v) glycerol in 0.05 M Tris, pH
7. Ethidium bromide staining solution contains 10 !l of a 10
mg/ml solution of ethidium bromide in 100 ml of deionized water.
Plasmid DNAPlasmid DNA (pUC118, Genbank accession no.
U07649.1, 3162 bp) [5] is prepared from 250-ml cultures of Escherichia coli that have been transformed with pUC118 and grown
as described in Sambrook and Russell [4]. After harvesting by
centrifugation, the plasmid DNA is obtained from the pellet of
bacteria by alkaline lysis with SDS and is purified by equilibrium
centrifugation in continuous cesium chloride-ethidium bromide
gradients. The purified plasmid is suspended in TE buffer to give
a final DNA concentration of #1 mg/ml and stored at 20 C. One
250-ml culture typically yields about 0.51 mg of DNA. Plasmid
DNA prepared in this way gives consistently better results for this
practical that does DNA prepared by resin-based chromatographic methods.
M13 DNAA culture of E. coli JM109 is infected with bacteriophage M13mp18 [6] (Genbank accession no. M77815.1, 7250

nt) as described in Sambrook and Russell [4]. The infected cells

are added to 250-ml batches of Luria Bertani medium in 2-liter
flasks and incubated at 37 C for 5 h with vigorous aeration.
Infected cells and phage particles are separated by centrifugation. Phage particles are precipitated by polyethylene glycol and
pelleted by centrifugation. Single-strand circular DNA is isolated
from this pellet by the procedures described in Sambrook and
Russell [4]. The covalently closed circular form of M13 DNA is
isolated from the bacterial pellet by the alkaline lysis procedure
also described in Sambrook and Russell [4], and purified by
equilibrium centrifugation in cesium chloride-ethidium bromide as
described above. Both forms of DNA are suspended at a suitable
concentration (0.51 mg/ml) in TE buffer and stored at 20 C.
EnzymesThe restriction endonuclease EcoRI and calf thymus DNA Topoisomerase I are obtained from Invitrogen. DNA
Topoisomerase I is diluted in topoisomerase storage buffer immediately prior to the experiment.
Agarose Gel SolutionThese are made from a 0.7% solution
of agarose in TAE buffer.

Sessions 1 and 2
Each group of students is provided with the following:
Horizontal gel box with comb (10 well, session 1; 20 well,
session 2). Suitable power supply.
100 ml of 0.7% agarose in 1" TAE buffer. (Melted and held
at 55 C in a water bath. Do not add ethidium bromide to the
agarose.)
1" TAE buffer sufficient for gel apparatus used.
100 ml of ethidium bromide staining solution.
1.5-ml microcentrifuge tubes.
Adjustable pipettors (220 !l, 20 200 !l, 0.1- 1.0 ml) with
appropriate tips.

Session 1
Each group of students is provided with the following:

70 !l of plasmid DNA (#0.51 mg/ml) in TE buffer.

25 ml of TE buffer.
Spectrophotometer capable of readings at 260 nm.
1-ml UV transparent cuvettes.

Session 2
Each group of students is provided with the following:
20 !l of plasmid DNA (200 !g/ml in TE buffer).
20 !l of M13 CCC DNA (200 !g/ml in TE buffer).
20 !l of M13 single-stranded circular DNA (200 !g/ml in TE
buffer).
10 !l of calf thymus DNA Topoisomerase I (Invitrogen) (1
unit/!l diluted in topoisomerase dilution buffer immediately
prior to the start of the practical).
15 !l of topoisomerase reaction buffer (10").
5 !l of EcoRI (1 unit/!l).
10 !l of React 3 buffer.
20 !l of a mixture of 1 kbp double-stranded DNA size standards (Invitrogen 1 kb ladder).
PROCEDURES

Session 1 and 2
Preparation of Agarose GelsThe 0.7% solution of agarose in TAE buffer is prepared and melted prior to the
practical period and held at 45 C in a water bath. Gels
with 10 (session 1) or 20 (session 2) sample wells are
required. The students pour the gels at the start of each
practical, before they begin the rest of the experiment.

30

BAMBED, Vol. 33, No. 1, pp. 28 33, 2005

Session 1

Quantification of Plasmid DNAThe students are told

the range within which the plasmid DNA concentration is
expected to be and are told that double-stranded DNA at
a concentration of 50 !g/ml has an absorbance at 260 nm
of 1.0.
After calculating the dilution required to give an absorbance of between 0.4 and 0.8 at 260 nm, students prepare
a 1-ml aliquot of this dilution in TE buffer in a 1.5-ml
microcentrifuge tube. After mixing, the absorbance is
measured at 260 and 280 nm against a blank of TE. The
actual concentration of their plasmid sample is then calculated, as is the A260/280 ratio. Students are asked to find
the A260/280 of highly-purified DNA by referring to the manuals (Sambrook and Russell [4]) available in the laboratory
and to compare their result with this.
Preparation of Serially Diluted Plasmid DNA Samples
Each student group prepares a series of dilutions of plasmid in which 10 !l contain the following amount of DNA: 1
!g, 500, 200, 100, 50, 20, 10, 5, 2, 1 ng. Dilutions are made
into TE buffer.
Loading Agarose Gels and ElectrophoresisThe combs
are removed from the gels and TAE buffer added to just
cover the agarose. Ten 2-!l drops of loading solution are
placed on a strip of parafilm on the bench adjacent to the
gel. A 220-!l pipettor is set to 10 !l and used to load the
gel, starting with the lowest concentration of DNA. Ten
microliters of each sample is mixed with a 2-!l drop of
loading solution, and the entire 12 !l is placed in one of the
wells of the agarose gel. This is repeated until all 10 wells
have been filled. If the samples are loaded in order from
the lowest to highest concentration, there is no need to
change the pipette tip between samples. Once all wells are
loaded, the lid is placed on the gel apparatus and the leads
are connected, checking that the polarity is correct. Students are encouraged to think about this by asking them
which direction they are expecting the deoxyribonucleic
acid in their samples to move. The power supply is adjusted to 100 V. The blue band of bromphenol blue will
move about 10 12 cm in 60 90 min. After turning off the
power, the leads are disconnected and the gel is carefully
transferred to a shallow container in which 5 !l of a solution of ethidium bromide (10 mg/ml) has been added to
250 ml of water. After staining for 30 60 min, the gel is
transferred to a container containing water only. The gel is
visualized and the results recorded with a suitable UV
transilluminator and documentation system.
Timing and Student ParticipationIn this practical, the
students take a long time with the calculations and the
preparation of the serially diluted plasmid DNA. In a 3-h
practical, they manage to get their gels loaded and running
but do not usually have time to stain and visualize the gels.
This is done for them by a technician, or by the person
supervising the laboratory, and the students pick up the
record of their experiment the next day.
SESSION 1 RESULTS AND DISCUSSION

The students are told that their plasmid preparation has

a DNA concentration of between 1 and 0.2 mg/ml and that
double-stranded DNA at a concentration of 50 !g/ml has

FIG. 1. Amounts of pUC118 plasmid DNA between 1 ng and

1 !g were applied to a 0.7% agarose gel in TAE buffer. A
potential of 100 V was applied until the bromphenol blue dye
band had moved #10 cm from the sample wells. The gel was
stained, visualized, and recorded as described under Materials
and Methods. The amounts of plasmid DNA are shown above
the appropriate lanes.

an absorbance at 260 nm (A260) of 1.0. An absorbance of

between 0.35 and 0.8 at 260 nm is required for an accurate
measurement and this means that a range of dilutions
between 1/5 and 1/50 might be appropriate. As they are
only provided with a total of 70 !l of plasmid they have to
make a decision where they balance the potential accuracy of their measurement with the amount of material they
have. Typically a 1/20 dilution to a total of 1 ml will give an
A260 of about 0.4 0.8, corresponding to a double-strand
DNA concentration of 400 800 !g/ml for the undiluted
sample. The A260/280 ratio is typically 1.8. The actual DNA
concentration is now used for the calculations required to
prepare a range of DNA solutions containing between 1 !g
and 1 ng in 10 !l of buffer, and these are used for agarose
gel electrophoresis.
A typical result for the agarose gel electrophoresis part
of the practical is shown in Fig. 1. About 5 ng of DNA in a
single band is the limit of detection with ethidium bromide
in agarose gels. Depending on the plasmid preparation,
the lanes with greater amounts of DNA should show two
well-separated bands, and there will often be three bands,
particularly if the plasmid DNA preparation has been subjected to several freeze-thaw cycles.
These results should be discussed with the class as a
group at the beginning of the next practical session. The
students need to be challenged by asking why a so-called
pure preparation of plasmid DNA has more than one band
and what the bands might be. They should reach the
conclusion that the multiple bands are due to different
topoisomers of the plasmid DNA but that there is not
sufficient information in the result shown in Fig. 1 to conclusively identify which of the potential topoisomers are
present. This discussion serves as a good introduction to
the second part of the practical where they treat plasmid
and bacteriophage M13 DNA with a restriction endonuclease and a topoisomerase.

Session 2
Treatment of Plasmid and M13 DNA with EcoRIThe
reaction mixtures shown in Table I below are set up in
microcentrifuge tubes placed on ice. All volumes shown
are microliters. Components are added in the order listed,
centrifuged briefly, mixed, centrifuged again, and all tubes

31
TABLE II
Protocol for DNA Topoisomerase I treatment of M13 DNA

TABLE I
Protocol for restriction endonuclease digestion of
M13 and plasmid DNA
Tube Number
H2O
10" React 3 buffer
M13 CCC DNA
M13 SS ! DNA
Plasmid DNA
Eco RI restriction endonuclease

E1

E2

E3

E4

E5

E6

6
1
3

5
1
3

6
1

5
1

6
1

5
1

3
1

are incubated at 37 C for 30 min. Ten microliters of DNA

gel loading solution is then added to each tube, mixed, and
placed on ice.
Topoisomerase I Treatment of Plasmid and M13 DNA
Four reaction mixtures are prepared as described in Table
II. All volumes are microliters. The tubes are kept on ice
and the components added in the order shown. The topoisomerase is not added until just prior to starting the
incubation.
Topoisomerase is added to the appropriate tubes, and
all tubes are mixed briefly and pulse-spun in the microcentrifuge. All reaction mixtures are incubated at 37 C for
30 min. Following the incubation 10 !l of gel loading
solution is added to each tube, mixed, and the tube placed
on ice.
Time Course Treatment of Plasmid DNA with Topoisomerase IFive microliters of gel loading dye is added to
each of six microcentrifuge tubes labeled Topo 0, Topo 1,
Topo 2, Topo 5, Topo 10, and Topo 20. All six tubes are
placed on ice.
The reaction mixture described below is prepared in a
single microcentrifuge tube. The components are added in
the order listed (Table III) and the tube kept on ice. Before
the DNA Topoisomerase I is added, the reaction is mixed
well and a 5-!l aliquot removed and mixed with the loading
dye in the Topo 0 tube.
Topoisomerase 1 is added to the remaining reaction
mixture. This is mixed well and incubated at 37 C. At 1, 2
5, 10, and 20 min, a 5-!l aliquot is removed and mixed with
the loading dye in the appropriately labeled tube and
placed on ice. Continue incubating the reaction tube.
Electrophoresis of All SamplesAfter removing the
comb from the agarose gel, 1" TAE electrophoresis buffer
is added to just cover the agarose. The DNA samples (10
!l of each) are placed in the wells of the gel as shown in
Fig. 2.
After all samples are loaded, the power supply is connected and adjusted to 100 V and the gel run until the
bromphenol blue dye band has migrated at least 10 cm.
The gel is stained with ethidium bromide and visualized as
described earlier.
SESSION 2 RESULTS AND DISCUSSION

The results of a typical experiment are shown in Fig. 2.

The main reason for failure in this section of the practical is
loss of activity of the Topoisomerase I. We have found that
it is advisable to obtain a fresh batch of enzyme each year
just before the practical is to be run. It is also essential that
the enzyme be diluted into topoisomerase storage buffer
(not topoisomerase reaction buffer) and that this is done

H2O
10" topoisomerase buffer
M13 CCC DNA
M13 SS ! DNA
DNA Topoisomerase I (1/10 dilution in
topoisomerase storage buffer)

T1

T2

T3

T4

4
1
5

3
1
5

4
1

3
1

5
1

TABLE III
Protocol for DNA Topoisomerase treatment of plasmid DNA
Reaction component

Volume (!l)

H2O
10" topoisomerase buffer
Plasmid DNA
DNA Topoisomerase I

35
5
5
5

FIG. 2. The reaction products of EcoRI and DNA Topoisomerase I treatment of M13mp18 and pUC118 DNA were
subject to agarose gel electrophoresis as described under
Material and Methods and the legend to Fig. 1. A 1-kbp
ladder (Invitrogen) acted as a size marker for linear duplex DNA
molecules.

just before the students require the enzyme.

The results obtained in the second part of the experiment and shown in Fig. 2 allow the interpretation of the
results of Fig. 1 from the first practical session. The EcoRI
digest of the plasmid shows that the two (or three) bands
of the uncut plasmid give rise to a single band following
digestion. The plasmid used was pUC118, which is 3.16
kbp, and the single band resulting from EcoRI digestion
has a mobility very close to the 3-kbp band of the marker
ladder. If the uncut plasmid DNA shows a third faint band
(just evident in lane E5 of Fig. 2), it runs with the same
mobility as the product of EcoRI digestion. This allows the
students to make the interpretation that the two major
bands shown in the uncut plasmid DNA of Figs. 1 and 2 are
probably supercoiled and relaxed circular DNA and that
the minor band of intermediate mobility is plasmid DNA
that has become linearized during plasmid isolation. Two
different plasmid preparations have been used to generate
the data shown in Figs. 1 and 2.
The natural assumption would be that the band with the
highest mobility would be the supercoiled plasmid because supercoiling compacts the circular plasmid. This

32

BAMBED, Vol. 33, No. 1, pp. 28 33, 2005

TABLE IV
Questions for students

FIG. 3. Log10 DNA fragment size is plotted against mobility

for the bands from the 1-kbp ladder shown in Fig. 2 (!). The
mobility of the linear products of EcoRI digestion of pUC118 and
M13mp18 have been plotted () and the apparent fragment size
calculated.

assumption is supported by the results of the time course

of the treatment of the plasmid with DNA Topoisomerase I
shown in lanes Topo 0 to Topo 20 in Fig. 2. Here the
intensity of the high-mobility band decreases with time of
treatment, and there is a corresponding increase in the
amount of the slower band. Evident at all time points after
zero time are the bands of a ladder of topoisomers, with
the distribution moving toward more relaxed forms as
digestion proceeds. This result supports the assumption
that the faster moving band in the untreated plasmid DNA
is highly supercoiled and the slower moving band is relaxed DNA.
The lanes that have the products of EcoRI and Topoisomerase I treatment of the single-stranded DNA (SS!) of
M13 (lanes E3, E4; T1, T2) show no change to the mobility
of the DNA band. This suggests that the restriction endonuclease and the topoisomerase have no activity toward
single-stranded DNA. The effect of EcoRI treatment of the
covalently closed double-stranded (ccc) M13 DNA should
also be commented on (lanes E1 and E2). Under the conditions of the experiment shown, the linear duplex DNA
has mobility slightly greater than that of the supercoiled
topoisomer in the adjacent lane. This emphasizes the point
that it is not possible to assign the topological form to the
bands seen following electrophoresis of uncut circular duplex DNA. In the case of pUC118, linear duplex DNA has
mobility between that of the fully supercoiled topoisomer
and the relaxed form.
The gel shown in Fig. 2 demonstrates a minor anomaly
that often occurs with this part of the experiment. The
lanes labeled Topo 0 and E5 contain identical DNA, and
the relaxed and supercoiled DNA should have the same
mobility in each lane. In our hands however, the bands in
the Topo 0 lane often have a slightly lower mobility than in
the E5 lane. We are not able to explain this anomaly
satisfactorily. The absence of the linear 3-kb band in the
Topo 0 lane is due to the somewhat lower amount of DNA
in this lane.
Students should also be required to make a plot of
mobility versus log10 of fragment size for the DNA ladder
used. An example derived from the data in Fig. 2 is shown
in Fig. 3. Preparing such a plot introduces the concept
that, for DNA molecules of the same topological form,
mobility is related to the size of the DNA molecule. The

1. What physiological manipulations of DNA in vivo require the

activity of DNA Topoisomerases?
2. In this experiment the gel electrophoresis was run without
ethidium bromide incorporated into the gel. When only linear
duplex DNA molecules are being run, it is common practice to
include ethidium bromide (2 !l of 10 mg/ml per 200 ml) in the
agarose solution when the gel is poured. This saves having to
stain the gel at the end and also means that the progress of the
electrophoresis can be followed during the run. Discuss why the
addition of ethidium bromide to the gel could have affected the
results of this experiment.
3. The topoisomerase used in this experiment is of type IB. What
does this mean in terms of the mechanism of action of the
enzyme? In the spread of topoisomers of pUC118, seen in the
time course part of the experiment, are the bands separated by
one supercoil? If the experiment were repeated with a type IA or
a type II topoisomerase, would the result be the same? Explain
your answer.
4. In the lane of untreated pUC118 plasmid DNA (lane E5, Fig. 2),
there is a small amount of DNA with a mobility corresponding to
the linear duplex DNA generated after digestion by EcoRI (lane
E6, Fig. 1). How is the DNA in this band generated in the
untreated plasmid DNA and what happens to the DNA in this
band when the preparation is treated with EcoRI?

DNA fragments in the 1-kb ladder are all linear duplex

molecules, and only DNA bands that are also due to linear
duplexes may be compared in this way.
Students need to decide which of the bands shown in
their topoisomerase and EcoRI digests can be used to
determine the size of the pUC118 and M13mp18 DNA
provided. They should realize the only bands that can be
compared in this way are the linear duplex molecules
resulting from EcoRI digestion of the double-stranded circular DNA from each DNA in lanes E2 and E6. The mobility
plot shown in Fig. 3 shows some deviation from linearity
that typically occurs when the gel is run quickly, as is the
case here. The mobility of the linear duplex bands of
pUC118 and M13mp18 are plotted on the figure, and the
corresponding fragment size is shown adjacent to the
y-axis. The value for pUC118 (3.13 kbp) is close to the
published value of 3.162 kbp. The value for M13mp18
(6.17 kbp) is considerably less than the published value of
7.25 kbp, probably due to the deviation from linearity in
this part of the curve.
Students can use a graphing program such as Microsoft
Excel to prepare the plot of log10 fragment size versus
mobility. We feel, however, that actually preparing and
plotting the data is a useful learning exercise. Log/linear
graph paper can also be provided, and the use of this is
also instructive.
DISCUSSION OF THEORETICAL ASPECTS OF EUKARYOTIC DNA
TOPOISOMERASE I ACTIVITY

Students should be encouraged to think about the reaction of type I DNA Topoisomerases, such as the one
used in this experiment, by the inclusion of appropriate
questions that will require them to consult the references
associated with the practical. Examples of these are provided in Table IV.
The enzyme used (DNA Topoisomerase I from calf thymus) is a eukaryotic topoisomerase and is a type IB enzyme [3]. Topoisomerases of this class relax both positive
and negative supercoils in a reaction that proceeds to

33
completion and does not require ATP. The type IB topoisomerases do not share sequence or structural homology
with any other topoisomerases and have a different reaction mechanism to the prokaryotic type IA enzymes. Type
I topoisomerases all change the topology of doublestranded DNA by creating a nick in one of the two strands
of the helix and passing the unbroken strand through the
gap created, followed by religation. They are all potentially
capable of changing the linking number by one, in contrast
to the type II topoisomerases that make a double-stranded
break and pass duplex DNA through the gap before religation. Consequently type II enzymes change the linking
number by 2.
Type IB toposiomerases form a covalent intermediate
where a tyrosine at the enzyme active site becomes covalently linked to the 3$-phosphate end of the cleaved
strand rather than to the 5$-phosphate end as in the type
IA enzymes. An additional point of difference between the
type IA and IB topoisomerases is in the nature of the
supercoil release by the two classes. Type IA enzymes
form a bridge across the broken DNA strand and allow
only a single-strand passage across the gap [7]. Type IA
enzymes change the linking number by one for each catalytic event. In contrast, the mechanism of action of type
IB topoisomerases has been proposed to involve a controlled rotation of the helix duplex about the uncut DNA
strand. This mechanism implies that the linking number
can change by greater than one for each breakage and
religation event. This has been investigated using the type
IB topoisomerase from Vaccinia [8]. In this study, an average change in linking number of five was shown. However,
this value can change with the conditions of the experiment as shown by Keller [9, 10] using human topisomerase
IB and SV40 DNA. This author demonstrated that lowering
the reaction temperature to 0 C increased the number of
relaxation intermediates.
The results shown in Fig. 2 support this conclusion.
Most covalently closed supercoiled DNA has a superhelical density of 0.06 [1]. A plasmid the size of pUC118
should therefore have 18 or 19 superhelical turns. The
most obvious intermediate bands shown in the time
course of topoisomerase treatment (lanes T0-T20 of Fig. 2)
number about 8. This is very similar to the result shown in
Fig. 5A of [8] for the relaxation of the related plasmid
pUC19 by Vaccinia DNA topoisomerase I, a type IB
topoisomerase.
Provided that the precautions noted are taken, the success rate for this practical is high. In the few instances
where there has been a total failure of the DNA Topoisomerase activity, this has always been due either to the

use of a batch of enzyme that has been stored for some

time, or the dilution of the enzyme too far in advance of
when it is required.
We find that the first session of the practical gives the
students a working knowledge of the sensitivity of
ethidium bromide-stained gels to the amount of DNA in a
band. It also serves to raise some questions that the
students have to consider in the second practical session.
The second session illustrates and consolidates their understanding of the activity of restriction endonucleases
and DNA Topoisomerases on various topological forms of
DNA.
In their report on this practical, students need to be
encouraged to extract as much information from their data
as possible. They should report not only what they observe, but also their interpretation of these observations.
For example, rather than merely stating that Topoisomerase I does not have any effect on M13 single-strand DNA
they should also realize that the concept of topoisomers,
as conventionally stated, only applies to duplex DNA molecules that are covalently closed or constrained in some
way.
AcknowledgmentsRobert Cleaver, Patricia McLenachan,
Sherralee Cleland, and Carole Flyger have all made valuable
contributions to setting up and maintaining this practical. We also
acknowledge the contribution of the students who have attempted this practical over the past 10 years and who have
provided valuable feedback.
REFERENCES
[1] J. D. Watson, T. A. Baker, S. P. Bell, A. Gann, M. Levine, R. Losick
(2004) DNA topology, Molecular Biology of the Gene, 5th Ed., pp.
111122, Pearson Education, New York.
[2] D. Voet, J. G. Voet (2004) Supercoiled DNA, Biochemistry, 3rd Ed.,
pp. 11221133, John Wiley & Sons, New York.
[3] J. J. Champoux (2001) DNA topoisomerases: Structure, function, and
mechanism, Annu. Rev. Biochem. 70, 369 413.
[4] J. Sambrook, D. W. Russell (2001) Molecular Cloning: A Laboratory
Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York.
[5] J. Viera, J. Messing (1987) Production of single-stranded plasmid
DNA, Methods in Enzymology (R. Wu, L. Grossman, eds.) Vol. 153,
pp. 111, Academic Press, San Diego, CA.
[6] C. Yanisch-Perron, J. Vieira, J. Messing (1985) Improved M13 phage
cloning vectors and host strains: Nucleotide sequences of the
M13mp18 and pUC19 vectors, Gene 33, 103119.
[7] J. J. Champoux (1990) Mechanistic aspects of type-I topoisomerases, DNA Topology and Its Biological Effects (N. R. Cozzarelli, J. C.
Wang, eds.) pp. 217242, Cold Spring Harbor Laboratory Press,
Plainview, NY.
[8] J. Stivers, T. Harris, A. Mildvan (1997) Vaccinia DNA topoisomerase I:
Evidence supporting a free rotation mechanism for DNA supercoil
relaxation, Biochemistry 36, 52125222.
[9] W. Keller (1975) Characterization of purified DNA-relaxing enzyme
from human tissue culture cells, Proc. Natl. Acad. Sci. U. S. A. 72,
2550 2554.
[10] W. Keller (1975) Determination of the number of superhelical turns in
simian virus 40 DNA by gel electrophoresis, Proc. Natl. Acad. Sci.
U. S. A. 72, 4876 4880.