Denaturing Gradient Gel Electrophoresis (DGGE)

Denaturing Gradient Gel Electrophoresis (DGGE)

❖ Denaturing Gradient Gel Electrophoresis (DGGE) is a molecular

biology technique used to analyze DNA fragments based on their
sequence variations.

❖ DNA samples that are the same size but have a different sequence
can take advantage of something called the melting temperature (Tm)
of the DNA.

❖ The T m is the temperature where the double-stranded DNA denatures

to form single-stranded DNA due to the loss of the hydrogen bonds
between the complementary base pairs.

❖ Different regions of a DNA molecule can have different T ms, and

these regions are called “melting domains.”

❖ It was first developed in the late 1980s by Päivi T. K. Mäkinen and

Petri T. K. Korpela, working at the University of Helsinki in Finland.
Their seminal paper describing DGGE was published in 1988.
❖ The DNA sequence within that domain determines the Tm of a melting domain, and a higher GC
content generally means a higher T m, as G-C pairs have three hydrogen bonds vs. the two bonds
found in an A-T pair.

❖ We can use this link between sequence and T m to look for a change in sequence between two
DNA samples of identical length by varying the denaturing conditions in different areas of a
polyacrylamide gel, causing different sequence to denature to varying points during
electrophoresis.

❖ DGGE exploits the principles of electrophoresis and denaturation to separate DNA fragments by
their sequence properties.

❖ Because even single base pair changes in DNA sequence can influence the T m of a melting
domain, DGGE can be a powerful technique for mutation detection.

❖ Its uses include identifying species in clinical microbiology, mutation detection in tissue samples,
genetic diversity, microbial communities, and environmental microbiology
 How does DGGE work?
❖ As the name suggests, DGGE uses a gradient of
denaturing strength along a polyacrylamide gel's
vertical or horizontal axis.

❖ PCR amplifies DNA samples from various sources

with identical primers and then electrophoresed
in this gel, similar to standard polyacrylamide gel
electrophoresis.

❖ As the DNA samples progress through the gel,

their melting domains will denature at different
points, depending on their T m.

❖ As they denature, these melting domains create

single-stranded branches that slow the migration
of the fragment within the gel matrix.
❖ This branching pattern is made possible by the “GC clamp,” a region of high GC content within the
PCR primers that prevents the two strands from completely dissociating and allows analysis of
practically any DNA sequence.

❖ In a mixed population of samples, this change in migration rate creates a banding pattern similar
to that seen for varying-size fragments in traditional gel electrophoresis.

❖ Comparison of these bands with a marker of known sequences can then be used to identify the
species present in the sample, and bands can be excised for DNA sequencing for confirmation.
Steps involved in DGGE:

1. Sample Collection and DNA Extraction:

❖ Collect the samples containing the DNA of interest (e.g., environmental samples, microbial cultures).
❖ Extract DNA from the samples using a suitable DNA extraction method.

2. PCR Amplification:

❖ Select a target gene or region for amplification. The 16S rRNA gene for bacteria and archaea, or the ITS region for
fungi, is commonly targeted in microbial community studies.
❖ Design and choose primers specific to the gene/region you want to study.
❖ These primers should have a GC clamp (a short sequence of GC bases) attached to one of the ends to facilitate DGGE
analysis.
❖ Perform PCR amplification to generate DNA fragments from the extracted DNA using the selected primers.

3. DGGE Gel Preparation:

❖ Prepare a polyacrylamide gel with a denaturing gradient. The denaturing gradient is created by mixing two solutions
with different concentrations of a denaturant (usually formamide and urea).
❖ Polymerize the gel and create a well for loading the PCR products.
4. Loading Samples and Electrophoresis:

❖ Mix the PCR products with a loading buffer and load them into the wells of the gel.
❖ Run electrophoresis. The DNA fragments will migrate through the gel based on their sequence and denaturing
properties. As the DNA fragments move through the denaturing gradient, they will denature at a position
corresponding to their GC content.

5. Staining and Visualization:

❖ Stain the gel with a DNA-specific dye (e.g., ethidium bromide) to visualize the DNA bands.
❖ Visualize the gel using UV light. You'll see bands representing different DNA fragments.

6. Analysis:

❖ Compare the band patterns between different samples. Similar band patterns indicate similar DNA sequences and,
thus, similar microbial communities.
❖ Excise bands of interest from the gel and extract DNA for further analysis (e.g., sequencing, cloning).
❖ If you want to identify specific microbial species, you can perform sequencing on the excised bands to determine their
DNA sequences.
❖ Remember, DGGE provides a snapshot of the microbial community based on DNA sequence similarities. It doesn't
provide quantitative information about the abundance of specific organisms.
❖ For more quantitative analyses, consider other methods like qPCR or metagenomic sequencing.
Instrumentation:

❖ To cast the DGGE gel, a particular piece of equipment

called a gradient mixer is used.

❖ this mixer mixes the two polyacrylamide solutions,

containing either high or low concentrations of the
denaturing agents Urea and Formamide, as the solutions
are poured into the gel casting apparatus.

❖ This mixing of solutions is carried out so that the bottom

of the gel has a high concentration of denaturing agent,
and the top of the gel has a low concentration, with a
gradual gradient between the two.

❖ This is called a “parallel” DGGE gel, as the denaturing

gradient runs parallel to the movement of samples in the
Casting a DGGE gel: Polyacrylamide solutions with high and gel matrix.
low concentrations of denaturing agents are mixed gradually
in a gradient mixer. This mixture is pumped into a casting
system, and the gel can polymerize between glass plates,
ready for electrophoresis.
❖ It is also possible to cast “perpendicular” DGGE gels where
the gradient runs from left to right.

❖ These gels are used to determine the best range of

denaturing agents for a specific sequence, and that range is
then used in the “parallel” DGGE described previously.

❖ Before the final experiment, one should optimize the

denaturant concentration for better separation using
perpendicular DGGE.

❖ Once the denaturant concentration is optimized, parallel

DGGE is performed to analyze the result.
 Application of DGGE:

1. Microbial Community Analysis:

❖ 16S rRNA Gene Analysis: DGGE is frequently used to study the diversity of bacterial communities in various
environments. By targeting the 16S rRNA gene (in bacteria) and running it through DGGE, scientists can generate a
fingerprint of the different species present in a sample.
❖ ITS Region Analysis: The Internal Transcribed Spacer (ITS) region is targeted for fungi. DGGE can be used to analyze
the diversity and composition of fungal communities in different habitats.

2. Mutation Detection:
DGGE can be used to identify mutations in genes. It’s instrumental in cases where known mutations affect the stability
of DNA, leading to differential migration patterns in the gel.

3. Single Nucleotide Polymorphism (SNP) Analysis:

DGGE can be used to detect single nucleotide changes in DNA. It’s beneficial in cases where the polymorphism alters
the denaturation behavior of the DNA fragment.

4. Species Identification:
DGGE can differentiate between closely related species or strains based on the unique patterns they generate in the
gel.
5. Environmental Studies:
DGGE can study microbial diversity in various environments, such as soil, water, or air samples. This can be
crucial in ecological and environmental research.

6. Genetic Disease Research:

DGGE can be used to detect genetic mutations associated with diseases. This can be helpful in diagnostic
settings or research studies aiming to understand the genetic basis of certain conditions.

7. Phylogenetic Studies:
By analyzing specific gene regions, DGGE can be used to construct phylogenetic trees, helping to understand
the evolutionary relationships between different organisms.

8. Drug Resistance Studies:

DGGE can be used to identify gene mutations associated with drug resistance in pathogens, helping develop
targeted therapies.

while DGGE is a powerful technique, it's important to interpret results in conjunction with other information and
sometimes to confirm findings with additional experiments or sequencing
 Example 1: Identifying microbial species in a mixed sample using DGGE
❖ Imagine a scenario in which there has been an outbreak of some bacterial infection in a livestock population. Treatment
requires a precise selection of antibiotics or other interventions depending on the causative agent.

❖ DGGE can be used to identify the species present in field samples to narrow down the possible causes and then
sequence the DNA fragments to identify the culprit.

❖ To start, isolate DNA from field samples and then amplify this DNA using PCR; since the target is to look for sequence
variations, the primers used for amplification are chosen from the conserved region of DNA that should be present in all
potential species/organisms. A common target is a region from the 16S ribosomal DNA sequence. GC clamp in one of the
primers to be included to create the high Tm region required to prevent complete dissociation of the DNA strands.

❖ Once PCR is complete and our gel is set, we load the products from different field samples in separate lanes of our DGGE
gel. The samples are electrophoresed, while the gel is kept at 60°C in a special warmed buffer tank to facilitate
denaturing. As the samples progress through the gel, DNA fragments of different species will denature and migrate
slower due to differences in their DNA sequence.

❖ After the completion of the gel electrophoresis, the gel is stained and imaged, and the pattern of distinct bands is
analyzed, each lane representing an individual species that was present in the original sample.

❖ We can run a set of known DNA samples alongside the experimental samples to quickly identify species in the mixture.
Individual bands can be excised from the gel and sent for DNA sequencing to confirm the presence of particular species,
and treatments/interventions can then be undertaken
 Example 2: Identifying mutations in cancer tissue samples
❖ In this example, we’ll cover how DGGE can screen for specific mutations in biopsy samples, such as single nucleotide
polymorphisms (SNPs).

❖ The development of breast cancer is influenced by two susceptibility genes, BRCA1 and BRCA2. Mutations in these genes
are over-represented in breast cancer patients compared with the general population.

❖ DGGE can rapidly screen large numbers of patients for mutations in BRCA1 and BRCA2 by comparing the patient samples
to a “wild-type,” a known unaltered sample.

❖ First, DNA is isolated from a biopsy sample from a patient. PCR is then performed using multiple primer sets in different
reaction mixtures to achieve complete coverage of the targeted genes.

❖ We use multiple PCR reactions as we are not just looking for differences in a single region to identify a species but the
entire gene coding region.

❖ Samples will migrate to specific points in the gel depending on their sequence, and the sensitivity of this method is such
that even a single base pair change can be detected.

❖ This method allows many patients to be rapidly screened for BRCA mutations without DNA sequencing.