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BIOCHEMISTRY
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
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OUTLINE OF THE PRACTICAL
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:
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.
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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
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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
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.
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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
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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