Denaturation Gradient Gel Electrophoresis

Denaturation gradient gel electrophoresis (DGGE) is a molecular biology technique for analyzing DNA fragments that can detect single-base variations or polymorphisms in DNA sequences. The principle of DGGE is that under different denaturing conditions, DNA fragments with different sequences will form different secondary structures in the gel, which will affect their migration rates. By comparing the DGGE patterns of different samples, the differences or similarities between DNA sequences can be found. DGGE has a wide range of applications in medical diagnosis, molecular evolution, microbial ecology, and other fields. It has the characteristics of high sensitivity, strong universality, simple operation, and low cost.

Principle and Steps of DGGE

Denaturation Gradient Gel Electrophoresis (DGGE) is a molecular biology technique for analyzing DNA fragments that can detect single-base variations or polymorphisms in DNA sequences. The principle of DGGE is that under different denaturing conditions, DNA fragments with different sequences will form different secondary structures in the gel, which will affect their migration rates. The principle of DGGE includes the following aspects:

  • Denaturing gradient: DGGE uses a gel containing denaturants (such as formaldehyde or urea), forming a gradient from low to high denaturation. Denaturants will break the hydrogen bonds and base stacking of DNA, causing DNA to partially or completely denature (unwind).
  • Tailed primers: To keep the DNA fragments in a single-stranded state under denaturing conditions, tailed primers are used in PCR amplification. Tailed primers are a special type of primer that have a GC-rich tail at the 5' end, which can form a stable hairpin structure. In this way, in the denaturing gradient, one end of the DNA fragment will remain double-stranded, while the other end will be single-stranded.
  • Migration stop point: When the DNA fragment migrates in the denaturing gradient, it will encounter a critical point that changes its secondary structure, called the migration stop point. At this point, the migration rate of the DNA fragment will significantly decrease or even stop. The migration stop point depends on the sequence and length of the DNA fragment, as well as the strength and direction of the denaturing gradient. DNA fragments with different sequences will have different migration stop points, forming different bands.

Table 1. The steps of DGGE

Step Main Content Purpose Precautions
Sample preparation Extract DNA from the target tissue or cell, and use tailed primers for PCR amplification. Choose the appropriate amplification region and primer pair to ensure that the amplified product can reflect the possible variations or polymorphisms in the DNA sequence. Obtain DNA fragments suitable for DGGE analysis. Avoid DNA contamination and degradation, optimize PCR conditions, and check amplification results.
Gel preparation Choose the appropriate denaturant and concentration, and prepare a polyacrylamide gel with a denaturing gradient. Depending on the need, a horizontal or vertical denaturing gradient can be prepared. Place the gel in a constant temperature water bath to stabilize. Make a denaturing gradient gel for separating DNA fragments. Strictly control the proportion and gradient of denaturants, avoid bubbles and cracks, and ensure gel quality and uniformity.
Electrophoresis analysis Load the amplified products onto the gel and perform electrophoresis at a constant voltage. According to the size and sequence of the DNA fragments, bands of different positions and intensities form in the denaturing gradient. Separate and display the migration stop points of DNA fragments in the denaturing gradient, reflecting their sequence differences or similarities. Choose the appropriate voltage and time, avoid overheating or overcooling, monitor current and temperature, and prevent bands from blurring or disappearing.
Staining detection Use a suitable dye to stain the gel, and use an imaging system to record the DGGE pattern. According to the position and pattern of the bands, analyze the differences or similarities between DNA sequences. Visualize and record DGGE results, providing a basis for subsequent data analysis and interpretation. Choose the appropriate dye and staining method, avoid over- or under-staining, adjust imaging parameters, and ensure image clarity and authenticity.

Application field of DGGE

DGGE has a wide range of applications in different fields. It can be used to analyze the differences or similarities between DNA sequences, to reveal gene function, phenotype, evolution, diversity, and so on. DGGE's applications include the following aspects:

  • Medical diagnosis: DGGE can be used to diagnose some genetic diseases caused by single-base variations or polymorphisms, such as cystic fibrosis, hereditary non-polyposis neurofibromatosis, hereditary breast cancer, and so on. DGGE can also be used to detect an individual's genetic background or drug metabolism ability to guide personalized treatment or drug dosage selection.
  • Molecular evolution: DGGE can be used to analyze the differences or similarities between DNA sequences of different species or populations to infer their phylogenetic relationships or evolutionary histories. DGGE can also be used to study gene selection pressure or adaptive variation to reveal gene function or phenotype in different environments.
  • Microbial ecology: DGGE can be used to analyze the structure and diversity of complex microbial communities, such as soil, water, and human or animal microbiota. DGGE can also be used to monitor the dynamic changes of microbial communities under different conditions, such as environmental pollution, bioremediation, disease occurrence, and so on.

References

  1. Mylvaganam S, et al. Denaturing gradient gel electrophoresis (DGGE) as a tool for identification of marine sulfate-reducing bacteria: comparison of 16S rDNA fragments obtained by DGGE and cloning based on sequence analysis. FEMS Microbiol Ecol. 2017 Jan;93(1):fiw209.
  2. Green SJ, et al. Denaturing gradient gel electrophoresis (DGGE) for microbial community analysis. In: Timmis KN, editor. Handbook of Hydrocarbon and Lipid Microbiology. Berlin, Heidelberg: Springer; 2010. p. 4137-4158.
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  5. Ohtomo R, et al.. PCR-denaturing Gradient Gel Electrophoresis as a Simple Identification Tool of Arbuscular Mycorrhizal Fungal Isolates. Microbes Environ. 2019 Dec 27;34(4):356-362.
  6. Resende PC, et al.. Standardization of denaturing gradient gel electrophoresis (DGGE) for discrimination of Victoria and Yamagata lineages of influenza B. J Virol Methods. 2012 Jan;179(1):212-6.
  7. M Weerasekera M, et al.. Denaturing gradient gel electrophoresis profiles of bacteria from the saliva of twenty four different individuals form clusters that showed no relationship to the yeasts present. Arch Oral Biol. 2017 Oct;82:6-10.
  8. Brito J, et al.. Characterization of eubacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) and Next Generation Sequencing (NGS) in a desulfurization biotrickling filter using progressive changes of nitrate and nitrite as final electron acceptors. N Biotechnol. 2020 Jul 25;57:67-75.
For research use only. Not intended for any clinical use.