DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE)

Imagine a scenario, where you have a patient with a life-threatening infection. Such a patient might require prompt
antimicrobial therapy. However, in clinical microbiology, there is this constant race against the clock in identifying
these disease-causing organisms so that an effective treatment can be provided. In fact, often, it is only after the
death of a patient that the disease-causing organism is identified. This is primarily because traditional culture
methods can be laborious, and in the case of mixed cultures, other analyses are required to identify
microorganisms at the species level. Taken together, the entire process can be time consuming, whereby
sometimes over a month is required to accurately identify the disease- causing organism.

It is therefore important to find ways to solve the problems that are found in extensive culture methods, and PCR-
DGGE (Polymerase Chain Reaction Denaturing Gradient Gel Electrophoresis) is one such way. In essence, PCR-
DGGE involves amplification of DNA by genus-specific primers that target 16S rDNA sequences and the
subsequent differentiation of this DNA on DGGE gel.

Introduction

DNA is a molecule responsible for preserving genetic information across species and across time. It consists of a
meaningful arrangement of chemicals called nucleotides that are symbolized by "A", "T", "C" and "G." These
arrangements tell us a story of each organism or individual, in that the code they produce, represent a detailed
instruction book for that organism or individual. Any change introduced in this sequence is called a mutation, and
whilst mutations occur randomly, many endure as an organism "acclimatizes" to a new environment. Hence,
observing and understanding these mutations would undoubtedly improve our understanding of residual and
transient organisms in a variety of environments when observation is difficult. For instance, this would be useful
for the monitoring of microbial populations, where culture dependant methods fall short. Denaturing Gradient Gel
Electrophoresis (DGGE) is such a technique that attempts to do this.

Denaturing gradient gel electrophoresis (DGGE) is a technique used for separating DNA fragments according to
their mobilities under increasingly denaturing conditions (usually increasing formamide/ urea concentrations). This
molecular biology approach is a fingerprinting methodology that has led to revolutionary changes in many of the
traditional routines used in assessing microbial populations.

How does it work?

First, DNA fragments from a sample containing multiple organisms are amplified using the PCR technique (to
learn more about PCR click the following link.) These amplified products generally entail sequences that are well
conserved from organism to organism - for example, sequences for the 16S rDNA are a common choice. This
collection of fragments is then subjected to the DGGE component of the procedure.

DGGE is a particular type of gel electrophoresis in which a constant heat (about 60°C) and an increasing
concentration of denaturing chemicals are used to force DNA molecules to unwind. A quick glimpse at
electrophoresis tells us that this is a separation technique based on the electrical charge, shape and molecular
weight of particulates such as DNA, proteins and RNA. In DGGE, DNA, which is negatively charged, is attracted
by the positive electrode and forced to migrate through the pores of a polyacrylamide gel. Once it reaches the
concentration of denaturing reagents at which it unwinds, it is said to have melted. This determines the melting
domains, which are defined as stretches of base pairs with an identical melting temperature. In other words, base
pairs formed by nucleotides A (adenine) and T (thymine), and those formed by C (cytosine) and G (guanine) are
chemically melted apart. Basically, what happens is that hydrogen bonding between the base pairs is broken by
the temperature and the increasing gradient of denaturing chemicals (urea and formamide). Any variation of DNA
sequences within these domains will result in different melting temperatures, thus causing different sequences to
migrate at different positions in the gel. This provides DGGE with the power to distinguish between mutated and
wild type sequences without prior knowledge of what these sequences are, justifying why this method is used to
detect mutations within closely related organisms. This separation is also aided considerably when a short
sequence of G's and C's (about 40 nucleotides), often-called GC-clamp, is attached to one end of the amplified
DNA products. This can be done by incorporation of this GC sequence into one of the primers used for amplifying

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the 16S rDNA fragments.

Figure 1: An example of a DGGE gel showing band compositions of various

populations samples, representative of complex microbial ecosystems. Each band
in each lane represents a 16S amplified product migrating to a unique position in the
gel, which melts in a sequence dependant manner.

Advantages

• Very sensitive to variations in DNA sequence;

• Allows simultaneous analysis of multiple samples;
• Useful method for monitoring shifts in community structure over time;
• Community profiles can be analyzed with cluster analysis:
• The use of universal primers allows the analyses of microbial communities without any prior knowledge
of the species;
• The fragments separated by DGGE can be excised, cloned and sequenced for identification;
• It is possible to identify constituents that represent only 1 % of the total community;
• DGGE can be applied to phylogenetic and functional genes.

Disadvantages

• DGGE analysis is rather time consuming;

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 • DGGE analysis suffers from the same drawbacks as all PCR-based community analysis techniques,
including biases from DNA extraction and amplification;
• The variation in 16S rRNA gene copy number in different microbes makes this technique only "semi-
quantitative";
• Microheterogeneity in rRNA encoding genes present in some species may result in multiple bands for a
single species and subsequently to an overestimation of community diversity;
• Heteroduplexes can cause biases to the observed diversity:
• No method for automated analyses currently available;
• Works well only with short fragments (<600 bp), thus limiting phylogenetic characterization;
• Gels of complex communities may look smeared due to the large number of bands;
• Band position does not provide reproducible taxonomic information;
• Results difficult to reproduce between gels and laboratories.

Applications
PCR-DGGE is classified as part of the new discipline of molecular microbial ecology. Microbial ecology aims at
studying interactions among microorganisms and between microorganisms and their environment. This involves
long-term study, which includes various and numerous environmental sample analysis. However, conventional
cloning, hybridisation and culture methods as mentioned above are not always practical for such investigations.
Moreover, these techniques do not provide any information on the dynamics of the microbial populations in
complex ecosystems and potential effects of environmental changes on such populations. Most importantly, these
methods require an extended knowledge of the microorganisms to develop adapted probes that target particular
individuals among diverse populations.
PCR-DGGE has the advantage of not requiring previous knowledge on microbial populations. It is a
fingerprinting approach that can generate a pattern of genetic diversity in complex microbial ecosystems such as
gastrointestinal tracts, soils, sediments, deep seas, rivers, hot springs and biofilms. A major advantage of this
method is its potential to visually profile and monitor changes occurring in various microbial communities, that are
undergoing different treatments or modifications. It is a rapid and efficient separation technique of same length
DNA sequences (amplified by PCR), which may vary as little as a single base pair modification.
Furthermore, PCR-DGGE is a flexible method that allows a unique combination of different approaches
for a more accurate identification of, for example, functional genes present in particular bacterial populations or
specific bacterial species by using hybridization or species-specific probes. This methodology can be utilized in
diverse subject areas such as clinical and environmental microbiology and food safety.
PCR-DGGE in clinical microbiology
Examples of DGGE applications in clinical microbiology abound. For instance, DGGE allowed the
identification of over 65 Mycoplasma species of human and veterinary origins in less than 24 hours. Mycoplasmas
are fastidious organisms that require many weeks to culture and other serological tests to be identified. They
cause various diseases associated with pneumonia, arthritis, conjunctivitis, infertility and abortion. This application
of PCR-DGGE could potentially allow considerable savings of time, life and treatment costs.
Another important achievement was that DGGE has been established to have a real potential in screening
large number of patients for rapid and reliable identification of deleterious changes in both breast cancer genes
BRCA1 and BRCA2. Hence PCR-DGGE allowed the detection of numerous mutations and revealed the existence
of unclassified variants that were not reported before. This method was also able to demonstrate that animals,
such as the Nile crocodile, baboon, red panda, wolf and Taiwan beauty snake, could also be infected by
Helicobacter species, bacteria suspected to be responsible for stomach ulcers.
PCR-DGGE in environmental microbiology and food safety
PCR-DGGE is also a useful tool in studying complex microbial communities such as the gastrointestinal
(GI) tract of food producing animals. Food producing animals are animals raised for milk, meat and egg production.
These animals can carry in their GI tract disease-causing organisms throughout the production chain to the retail
market and from the retail market to the consumer's dinner table (10). It is therefore very important to elucidate
the exact microbial populations of food producing animals' GI tract. This will allow a better control of the shedding
of deadly bacteria strains into manure which is used as fertilizer for produce such as fruits and vegetables.

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 The recent outbreak of E. coli on spinach in California is a painful recall that even vegetarians are not safe
from the damages caused by a degraded health condition of food producing animals. The need for rapid and
accurate methods for screening of total microbial populations in complex ecosystems is more evident than ever.
PCR-DGGE has proved to be a powerful tool in assessing total gut microbial populations and was also used to
detect previously unknown bacteria species in the GI tract of animals. Understanding the relationship between the
host and the disease-causing organisms will certainly assist us in defining efficient pathogens control measures.
This is of paramount importance in food safety and food processing where quality control and assurance programs
necessitate proficient methods to discontinue the transmission cycle of life- threatening microbes.
Last but not least, PCR-DGGE was used to examine complex ecosystems such as river, seas, soils and
deep-sea hydrothermal vent. Understanding complex microbial populations would certainly help us in rapid
decision-making with regard to adequate treatment and other major interventions aiming at making the world a
better and safer place to live.
DNA-based DGGE as a monitoring tool

It is especially suitable for samples with few species and gives complementary information on species diversity
when combined with other methods. It is also well suited for analysing community changes over time.

• Very suitable technique for the identification of novel or unknown organisms. The detection sensitivity is
limited due to PCR and primer artefacts, usually the most abundant species are detected.

• Easy to perform
• The most abundant species can be readily detected.
• Limited information about the abundance of detected species and no information about the activity of the
detected species.

• Long analysis time (several days) that makes it difficult to use it as an "on-line" tool.
Current use in bioleaching studies
DGGE has been used to elucidate and characterize microbial populations in many bioleaching environments,
including bioreactors (Kinnunen and Puhakka 2004), acidic mining-impacted environments (Gonzalez-Toril et al.
2003), and bioleaching heaps (Hawkes et al. 2004).

Conclusion

DGGE is undeniably a valuable approach in screening complex ecosystems on a large scale and in analyzing
various environmental samples in a reduced amount of time. Using this technique, diagnosis of emerging
infections could become easier and faster, and identification of uncultivable pathogens can also now be facilitated.
Although there are limitations, DGGE is an interesting and unique approach that bridges many molecular biology
tools together, and its limitations are primarily attributable to the fact that it is still a relatively new technique. If
improved,

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