Application of DGGE Technology in Microbial Ecology

The main principle of DGGE technology is shown in Figure 1. Different denaturant concentrations are required for DNA double-strand melting of base sequences. Once they are melted, the electrophoretic behavior in polyacrylamide gel will occur greatly. Variety. Therefore, the isometric DNA fragments obtained by PCR amplification are electrophoresed in a gel containing a denaturant gradient, and DNA fragments having different sequences are denatured at respective denaturant concentrations, and spatial configuration changes occur, resulting in electrophoresis. The rate of drastic drop is such that it stays in the corresponding denaturant gradient gel, which after staining appears as separate bands on the gel, each strip representing a specific sequence of DNA fragments. Strips that remain in the same position in different lanes are generally considered to have the same DNA sequence. This technique can resolve sequence differences with fragments of the same size and can be used to detect changes in single bases, genetic diversity of microbial communities, and polymorphisms in PCR amplified DNA fragments.


Figure 1 DGGE reaction principle In order to improve the detection sensitivity of DGGE, a GC-rich region (GC-Clamp, usually 30 to 50 nucleotides in length) is usually designed to be added to the 5' end of one primer. The complete melting of DNA increases the detection rate of different bases, so that sequences separated by one base can also be resolved.

2 Technical steps
The main operation process of this technology is as follows: (1) total DNA extraction of samples; (2) PCR amplification of 16SrDNA fragments of samples; (3) optimization and analysis of DGGE conditions; (4) subsequent processing such as tapping sequencing.

3 DGGE application prospects
Since Handelsman et al. proposed metagenomics (Entagenomics) in 1998, the use of modern genomics techniques to study microbial communities in natural or artificial environments has become a hot trend. At present, metagenomics has been applied to the study of marine and soil microbial communities. Functional genes derived from uncultured microorganisms in the environment, and even genomics research, broaden the field of research on microbial communities. DGGE technology can monitor specific functional genes and their expression studies in complex environments, providing favorable information and genetic screening solutions for metagenomics research. In addition, the nucleic acid sequences obtained in DGGE analysis can be further applied to fluorescence in situ hybridization, DNA microarray and real-time PCR (PCR) to contribute to the rapid detection of specific populations.

DGGE is currently a widely accepted molecular biology tool for environmental microbiology complex and behavioral research in environmental microbiology. The technology is reliable, repeatable, fast and easy to operate. DGGE, which combines PCR amplification of marker genes or their transcripts (rRNA and mRNA), directly displays the dominant components of the microbial community. Because it can analyze multiple samples at the same time, it is very suitable for investigating the spatiotemporal changes of microbial communities, and it is possible to identify the members of the community by sequence analysis of the cut bands or by hybridization with unique probes. Coupled with the theoretical background of the role of this technology, such as the thermodynamics of the melting behavior of double-stranded DNA in solution. Use DGGE to understand the fate of a microbial community or some indicator microorganism after the environment is disturbed. Conventional use of functional genes as molecular markers can be used to identify closely related but ecologically distinct populations. End-labeled fluorescent PCR products and (intra-lane) standards can be used to detect rare population members and facilitate accurate comparisons between samples and samples. Double gradients (in combination with polyacrylamide gradients and denaturant gradients) can be obtained by DGGE analysis. Better resolution. In order to reduce the bias and limitations of various technologies, combined with PCR-DGGE and other molecular biology techniques and microbiological methods will more reveal the structure and function of microbial communities.

Although DGGE electrophoresis has many advantages in studying community dynamics and diversity, it does not provide information on metabolic activity, bacterial count, and gene expression levels. Therefore, it must be combined with other technologies to make up for the shortcomings. Both Biolog and enzymatic analysis can provide metabolic activity characteristics of the community. In addition, fluorescence in situ hybridization can provide relative information on bacteria, and in situ hybridization of stable isotope probes can provide metabolic information and obtain community structure and functional relationships. The 16S rDNA or gene library is also a method for analyzing the relative numbers of different populations. The number of bacteria and gene expression levels in the community can be quantified by real-time PCR amplification of 16S rRNA or functional genes of specific species. Therefore, combined with other molecular biology techniques, the efficacy of DGGE technology can be further enhanced to better serve microbial community structure and functional analysis. (Article Source:

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