GREAT AT SMALL THINGS

Denaturing gradient gel electrophoresis (DGGE) at BCCM/LMG

Denaturing Gradient Gel Electrophoresis (DGGE) is a PCR-based microbial community fingerprinting technique that generates band profiles representing the sequence diversity of a target gene (e.g. 16S rRNA) within the ecosystem present in a microbial sample. Its main steps involve (i) extraction of total DNA from the sample, (ii) PCR amplification of an internal fragment of the target gene, (iii) sequence-dependent separation of the PCR amplicons along a chemical gradient by DGGE, and (iv) visualization and processing of DGGE fingerprints. The generated population fingerprints provide information on the microbial (genetic) diversity and community dynamics within/between the sampled ecosystems.

At BCCM/LMG, DGGE is offered as a customer service for (i) fast characterization of microbial content in mixed cultures or synthetic communities (e.g. product quality control), (ii) evaluation of treatments or interventions (e.g. pro/pre/synbiotics, antibiotics, bioactive compounds, etc.) on the diversity and stability of the autochthonous ecosystem in the sample(s), (iii) monitoring of temporal stability during microbial succession processes (e.g. fermentation, degradation, spoilage, etc.), and (iv) comparison of microbial diversity and species richness between conventional culturing and culture-independent approaches such as DGGE. In addition, BCCM/LMG also offers extraction, purification and sequencing of specific DGGE bands of interest for taxonomic purposes.

BCCM/LMG has expertise in DGGE analysis of several sample types, including fecal samples and colon biopsies, various food samples (e.g. dairy products, sourdough), probiotic products and lyophilized strain mixes. Upon request, and if sample numbers are sufficient, we can also optimize DNA extraction protocols for other sample types.

DGGE profiles are generated using the DCode Universal Mutation Detection System (Bio-Rad).

The service report includes detailed technical description of the protocol and visual interpretation of digitized profiles. On request, we can also perform clustering and richness analysis of DGGE fingerprints using BioNumerics v7.0 software, and compare these with previous or future samples (or isolates thereof) that the customer has/will send us.

 

Literature

Hamet, M.F., A. Londero, M. Medrano, E. Vercammen, K. Van Hoorde, G.L. Garrote,, G. Huys, P. Vandamme & A.G. Abraham. 2013. Application of culture-dependent and culture-independent methods for the identification of Lactobacillus kefiranofaciens in microbial consortia present in kefir grains. Food Microbiology 36:327-334. doi:10.1016/j.fm.2013.06.022

Huys, G., T. Vanhoutte & P. Vandamme. 2008. Application of sequence-dependent electrophoresis fingerprinting in exploring biodiversity and population dynamics of human intestinal microbiota: what can be revealed? Interdisciplinary Perspectives on Infectious Diseases - Special issue: The Human Microbiome and Infectious Diseases: Beyond Koch vol. 2008, Article ID 597603, 26 pages, 2008. http://dx.doi.org/10.1155/2008/597603

Joossens, M., G. Huys, K. Van Steen, M. Cnockaert, S. Vermeire, P. Rutgeerts, K. Verbeke, P. Vandamme & V. De Preter. 2011. High-throughput method for comparative analysis of denaturing gradient gel electrophoresis profiles from human fecal samples reveals significant increase in two bifidobacterial species after inulin-type prebiotic intake. FEMS Microbiology Ecology 75:343-349. http://dx.doi.org/10.1111/j.1574-6941.2010.01008.x