Denaturing Gradient Gel Electrophoresis (DGGE)
What is it?
Denaturing Gradient Gel Electrophoresis (DGGE) is a genetic fingerprinting technique used
to separate multiple DNA sequences according to their mobility in increasingly denaturing
conditions.
How does it work?
Firstly, DNA is extracted from a sample. The bacterial DNA is then amplified using the
Polymerase Chain Reaction (PCR). PCR products of the same size but differing nucleotide
sequence composition can then be separated using DGGE.
Each PCR product is added to a lane of a polyacrylamide gel containing a linear gradient of
DNA denaturants (urea and formamide). Double-stranded DNA is partially denatured as it
migrates through the gel. Separation of differing nucleotide sequences within a sample occurs
due to the decreased mobility of partially melted double-stranded DNA molecules when
migrating through a solution/gel under the influence of an applied electric field.
DNA partially denatures in discrete regions called melting domains.
Melting domains are stretches of base-pairs with an identical melting
temperature. The melting temperature of each domain is sequence
specific i.e. each unique nucleotide sequence melts differently.
Once a domain reaches its melting temperature, the migration
of that domain halts. This allows domains with different
nucleotide sequences to stop migrating at different positions
in the gel. Separated is therefore based on the difference in
melting behaviour of individual melting domains as the
DNA denatures.
A GC-rich sequence known as the GC clamp is attached to
one side of a DNA fragment during the PCR. The GC clamp
prevents two DNA strands from completely disassociating
and therefore results in nearly 100% detection of nucleotide
sequence variation.
DNA bands on a DGGE gel can be visualised using staining
dyes such as ethidium bromide or SYBR Gold.
Why is it used?
DNA extraction
PCR
DGGE
Flow diagram showing an example of the
different steps in analysis of microbial
community structure using DGGE. DNA is
extracted from an environmental sample and
the 16S rRNA encoding genes of the
bacterial DNA amplified via PCR. Different
bacterial DNA sequences are then separated
using DGGE. The DGGE bands can then be
excised to determine phylogenetic
affiliations.
DGGE is a rapid, reliable, reproducible and inexpensive
technique. One of the strongest aspects of DGGE is its ability to allow simultaneous analysis
of multiple samples taken from communities at different time points. Community
composition, and diversity can be monitored as well as seasonal and environmental changes
or fluctuations over time.
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An additional feature of DGGE is the possibility to identify community members by
sequencing excised bands. Since each new DNA band in a profile is likely to be derived from
one (or very few) phylogenetically distinct populations, an estimate of species number and
abundance based on the number and intensity of amplified fragments in a profile can also be
obtained.
Does it have any limitations?
DGGE suffers from some general potential biases. These are linked to sample type, handling
and storage as well as biases in the PCR technique and inefficiencies in DNA extraction
methods.
DGGE also has specific limitations. These include: limited sensitivity of detection for some
rare community members (only predominant species in a community are displayed) and the
co-migration of DNA fragments with different sequences.
There can also be problems separating relatively small DNA fragments, and the production of
molecules produced by different rRNA operons of the same organism. Heteroduplex
molecules can also form when strands of DNA from two different PCR products re-anneal.
However, some of these limitations can be improved or overcome. For example, limited
sensitivity issues can be improved using hybridisation analysis and the formation of
heteroduplex molecules can be reduced by optimising PCR conditions.
Despite the rapid development of high-throughput sequence analyses techniques, DGGE
remains the optimal method to provide an accurate and rapid overview of community
composition from which more detailed sequence analyses can be completed.
Further reading
Gerard, M. (1999). DGGE/TGGE a method for identifying genes from natural ecosystems.
Current Opinion in Microbiology 2, 317-322.
Muyzer, G. and Schäfer, H. (2001). Denaturing Gradient Gel Electrophoresis in Marine
Microbial Ecology. Methods in Micro 30, 425-468.
Muyzer, G. and Smalla, K. (1998). Application of denaturing gradient gel electrophoresis
(DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology.
Antonie van Leeuwenhoek 73, 127-141.
Øvreås, L., Forney, L., Daae, F.L. and Torsvik, V. (1997). Distribution of bacterioplankton
in meromictic Lake Saelenvannet, as determined by denaturing gradient gel
electrophoresis of PCR-amplified gene fragments coding for 16S rRNA. App and Env
Mic 63, 3367-3373.
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