Denaturing Gradient Gel Electrophoresis

In this article we will cover the theory behind denaturing gradient gel electrophoresis (DGGE), as well as how to cast and run a DGGE gel using our DGGE system. To follow this article, you will need a basic understanding of gel electrophoresis the structure of DNA and PCR (polymerase chain reaction). You can see an introduction to these topics on our applications page.

Contents

Introduction

With a routine electrophoresis experiment, we use agarose or polyacrylamide gel to separate DNA or Protein according to its size/molecular weight. But what if we want to look at DNA samples that are the same size, but have a different sequence? We can take advantage of something called the melting temperature (Tm) of the DNA. The Tm is the temperature where the double stranded DNA denatures to form single stranded DNA, due to the loss of the hydrogen bonds between the complimentary base pairs.

Different regions of a DNA molecule can have different Tms and these regions are called “melting domains”. The Tm of a melting domain is determined by the DNA sequence within that domain, and a higher GC content generally means a higher Tm, as G-C pairs have 3 hydrogen bonds vs the 2 bonds found in an A-T pair as shown in figure 1. We can use this link between sequence and Tm 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 at different points during electrophoresis. This technique is called denaturing gradient gel electrophoresis and is explained in more detail below.

GC Content Graph
Figure 1: Melting temperature of DNA is correlated to the % content of G-C bonds in the sequence. The higher the number of GC bonds, the higher the melting temperature.

Because even single base pair changes in DNA sequence can influence the Tm of a melting domain, DGGE can be a powerful technique for mutation detection. Its uses include the identification of species in clinical microbiology, mutation detection in tissue samples and environmental microbiology.

How does DGGE work?

As you would expect from the name, DGGE works by using a gradient of denaturing strength along either the vertical or horizontal axis of a polyacrylamide gel. DNA samples from various sources are amplified by PCR 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 in the gel, depending on their Tm. 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. The whole process is summarised in figure 2. To help explain the process even further, lets go through some practical examples.

Figure 2: Overview of DGGE theory. The separation of DNA based on sequence is caused by the different in denaturing agent concentration at the top and bottom of the gel, which changes the Tm of the DNA fragment.
Figure 2: Overview of DGGE theory. The separation of DNA based on sequence is caused by the different in denaturing agent concentration at the top and bottom of the gel, which changes the Tm of the DNA fragment.

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 precise selection of antibiotics or other interventions depending on the causative agent. We can use DGGE to identify the species present from field samples to narrow down the possible causes and then sequence the DNA fragments to definitively identify the culprit.

To start, we isolate DNA from field samples from infected individuals. This DNA is then amplified by PCR. Since we are looking for sequence variations, we will amplify using primers for a conserved region of DNA that should be present in all potential species/organisms present. A common target is a region from the 16S ribosomal DNA sequence. We will also include a GC clamp in one of our primers, to create the high Tm region required to prevent complete dissociation of the DNA strands.

Next, we cast our DGGE gel. Using a special piece of equipment called a gradient mixer, we can mix two solutions of polyacrylamide, 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 in such a way 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 gel matrix. See figure 3 for an overview of the DGGE casting method. It is also possible to cast “perpendicular” DGGE gels in which 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.

Figure 3: Casting a DGGE gel. Polyacrylamide solutions with high and low concentrations of denaturing agents are mixed gradually in a gradi-ent mixer. This mixture is pumped to a casting system when the gel can polymerise between glass plates, ready for electrophoresis.
Figure 3: Casting a DGGE gel. Polyacrylamide solutions with high and low concentrations of denaturing agents are mixed gradually in a gradi-ent mixer. This mixture is pumped to a casting system when the gel can polymerise between glass plates, ready for electrophoresis.

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, different species DNA fragments will denature and begin to migrate slower, due to differences in their DNA sequence.

Once the gel run is complete and the gel is stained and imaged, we see a pattern of distinct bands, each 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 present 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.

How to use the cleaver Scientific DGGE system to cast a Denaturing Gradient Gel

Example 2: Identifying mutations in cancer tissue samples

Example 1 focused on how we can use DGGE to identify species in a mixed population based on their DNA sequence. In this example we’ll cover how DGGE can be used to screen for specific mutations in biopsy samples, such as single nucleotide polymorphisms (SNPs).

The development of breast cancer is influence by two susceptibility genes, BRCA1 and BRCA2. Mutations in these genes have be found to be over-represented in breast cancer patients compared with the general population and so screening for these mutations has been identified as an early intervention for breast cancer prevention and treatment. DGGE can be used to 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.

The process is similar to that for screening microbiological samples outlined above. First DNA is isolated, this time 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 coding region of the gene.

Gel casting remains the same as in example 1, although the concentration of the high and low solutions may change depending on the sequence to be analysed. The PCR products are run on this gel, and compared with products from control samples, either “wild-type” or known variants. 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. The presence of specific mutations can be further confirmed by “heteroduplex analysis” in which two sample, one unknown and one known, are denatured and mixed prior to DGGE. When the samples are identical, the bands present on the gel remain the same, but where mismatched occur, new bands will appear that represent a change in Tm due to the formation of a heteroduplex between the two different strands.

Using this method, many patients can be rapidly screen for BRCA mutations without the need for DNA sequencing.

Figure 4: Example of a DGGE gel run on the Cleaver Scientific DGGE system.
Figure 4: Example of a DGGE gel run on the Cleaver Scientific DGGE system.

Equipment for DGGE

To carry out DGGE you will need some equipment that is commonly found in molecular biology laboratories, and some that is not. Below we’ll discuss the equipment required and the available equipment from Cleaver Scientific.

Since the first stage in DGGE is most often PCR, you will need a thermal cycler, to produce the PCR products to be run on the gel. The Cleaver Scientific GTC96S thermal cycler is perfect for this application.

To cast and run DGGE gels, you will need a gradient mixer, magnetic stirrer and optionally a peristaltic pump to pump the acrylamide solutions from the gradient mixer to the casting system. You will also need a large vertical electrophoresis system with a heated buffer chamber. The Cleaver Scientific DGGE system is perfect for this application and can be purchased as a kit, with all the necessary equipment including gradient mixer, stirrer and peristaltic pump. For more information on this equipment, visit the product pages below.

Figure 5: The Cleaver Scientific DGGE System.
Figure 5: The Cleaver Scientific DGGE System.

Finally, to image and document your gel, you will require a gel documentation system. For an economical option, consider the Cleaver Scientific microDOC, which can image the 20 x 20 cm gels produces in our DGGE system. For more demanding gel imaging, our complete range of gel documentation systems is available including options with touch screens, or the possibility to upgrade to chemiluminescence. Browse our full range below.

To analyse your imaged gels, you can use our intuitive gel analysis software, CLIQS, by TotalLAB. This software automatically detects bands and can create dendrograms from each lane to identify mutants and variants. With CLIQS 1D PRO you can also compare lanes between different gels for more advanced analysis.

For any questions about getting your lab set up to run DGGE, or about our DGGE equipment, contact our sales team on sales@cleaverscientific.com

References

  1. McAuliffe, L., Ellis, R.J., Lawes, J.R., Ayling, R.D. and Nicholas, R.A., 2005. 16S rDNA PCR and denaturing gradient gel electrophoresis; a single generic test for detecting and differentiating Mycoplasma species. Journal of Medical Microbiology, 54(8), pp.731-739.
  2. Hout, A.H.V.D., Ouweland, A.M.V.D., Luijt, R.B.V.D., Gille, H.J., Bodmer, D., Brüggenwirth, H., Mulder, I.M., Vlies, P.V.D., Elfferich, P., Huisman, M.T. and Berge, A.M.T., 2006. A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting. Human mutation, 27(7), pp. 654-666.