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4928-4929
Nucleic Acids Research, 1995, Vol. 23, No. 23
© 1995 Oxford University Press
A simple and rapid electrophoresis method to detect
sequence variation in PCR-amplified DNA fragments
Cathrin Wawer, Hermann Riiggeberg1, Georg Meyer12-* and Gerard Muyzer
Max-Planck-lnstitute for Marine Microbiology and 1Hanse Analytik GmbH, FahrenheitstraBe 1, D-28359 Bremen,
Germany and 2University of Bremen, FB2, Postfach 330440, D-28334 Bremen, Germany
Received September 19, 1995; Revised and Accepted October 31, 1995
Several methods have been described to detect sequence variation
in different, but equal-sized DNA fragments. Among them are
denaturing gradient gel electrophoresis (DGGE) (1), temperature
gradient gel electrophoresis (TGGE) (2), and single-strand
conformational polymorphism (SSCP) (3). All these methods
utilize polyacrylamide gels, need special equipment, and require
pre-experiments to determine the optimal electrophoretic conditions. Another limitation is that only relatively small DNA
fragments (up to 500 bp) can be separated with these methods.
Here we present a simple, and rapid electrophoresis method
which uses agarose gels, does not need special equipment and
allows larger DNA fragments to be separated. Electrophoresis is
performed in agarose gels containing the DNA ligand bisbenzimide, to which long chains of polyethylene glycol (PEG) 6000
are covalently coupled (4). The dye bisbenzimide binds preferentially to A+T sequence motifs in the DNA (5). Therefore, being
loaded with the long PEG chains, the A+T-rich DNA sequences
are retarded in the gel relative to the sequences which are low in
A+T content, and so separation is achieved. The principle of this
method has been known for 15 years (4), but has only been used
incidentally (6).
We have extended the use of this method in separating
PCR-amplified DNA fragments of the [NiFe]hydrogenase gene
from different sulfate-reducing bacteria. Two sets of primers were
used to amplify fragments of -440 and 1440 bp, respectively (7).
The PCR products were electrophoresed in agarose gels with and
without the bisbenzimide/PEG dye in parallel. Figures 1A and 2A
clearly show that PCR products obtained from different bacteria
are difficult to separate from each other in agarose gels lacking the
bisbenzimide/PEG, but can in gels containing the dye (Figs 1B
and 2B). Only partial sequence data of the amplified fragments
are available (Wawer and Muyzer, unpublished data). However
for Desulfovibrio gigas (9) and two other Desulfovibrio strains
(10,11) complete sequences have been published. These data,
with sequence similarities ranging between 70 and 90%, indicate
that differences in AT content of <1% have been resolved.
A better separation, especially for longer DNA fragments, can
be achieved by extending the electrophoresis time. However,
because the bisbenzimide/PEG dye slowly migrates in the
opposite direction to the DNA, a reservoir of gel containing the
DNA ligand in front of the fragments will be necessary.
Furthermore, immersion of the gels with electrophoresis buffer,
which is common in agarose gel electrophoresis, should be avoided
to prevent diffusion of the dye out of the gel. For these reasons,
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Figure 1. (A) PCR fragments (440 bp long) corresponding to the [NiFe]
hydrogenase gene from Desulfovibrio desulfiiricans DSM 1926 (lane 1),
D.vulgaris DSM 644 (lane 2), D.gigas DSM 1382 (lane 3), D.baculaius DSM
2555 (lane 4) and an equimolar mixture thereof (lane 5) are run in an agarose
gel containing 3.5% (w/v) agarose (low endoendosmosis, FMC) in 25 mM
EDTA, pH 5.9, with U = 3 V/cm for 3 h. The fragments cannot be separated
from each other, because separation is according to length. (Lane M: DNA size
standard pUCI8 HpaU). (B) Identical PCR fragments, but electrophoresed in
an agarose gel containing bisbenzimide/PEG (Hanse Analytik GmbH, Bremen,
Germany). The dye has been added to the agarose solution at a temperature of
60°C, and at a concentration of 0.025 O.D. units (340 nm) per ml. Separation
of the fragments is achieved based on their difference in A+T content.
the use of a Studier-type of electrophoresis apparatus (8) is
recommended.
Apart from the ease in preparing agarose gels, blotting and
DNA extraction from these gels is also easier to perform than
from polyacrylamide gels.
It might be envisioned that this electrophoresis method has
great potential for applied as well as basic research. The potential
use is in the characterization of microorganisms. In this study, it
is shown that closely related strains can be discriminated by using
bisbenzimide/PEG electrophoresis of PCR-amplified DNA fragments. By choosing the appropriate primer system, the method
will contribute to a rapid characterization of microorganisms in
mixed populations. We expect this to be a useful tool to screen
bacterial cultures for contaminants or new strains and to determine
and monitor the microbial diversity in environmental samples.
Furthermore, the method may be used to detect mutated genes.
Miiller et al. (4) postulated the possibility of detecting point
mutations in DNA fragments of 200-300 bp. As has been
*To whom correspondence should be addressed at: Hanse Analytik GmbH, FahrenheitstraBe I, D-28359 Bremen, Germany
Nucleic Acids Research, 1995, Vol. 23, No. 23 4929
REFERENCES
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Figure 2. The same conditions as described in the legend to Figure I, but with
longer PCR fragments (1440 bp) of the [NiFe] hydrogenase gene, and on a 2%
(w/v) agarose gel. (Lane M: DNA size standard X ///ndlll/EcoRI)
demonstrated for DGGE, TGGE and SSCP, the resolution of this
method is still to be determined empirically.
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