Optimization of the DGGE band identification method
Language English Country United States Media print-electronic
Document type Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't
- MeSH
- Rumen microbiology MeSH
- Bacteria classification genetics isolation & purification MeSH
- Denaturing Gradient Gel Electrophoresis methods MeSH
- DNA, Bacterial genetics MeSH
- Phylogeny MeSH
- Bacterial Typing Techniques methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Evaluation Study MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- DNA, Bacterial MeSH
Denaturant gradient gel electrophoresis (DGGE) enables insight into the diversity of the studied microbial communities on the basis of separation of PCR amplification products according to their nucleotide sequence composition. However, the success of the method is accompanied by the inherent appearance of various sequence artifacts that bias the impression of community structure by generating additional bands representing no virtual microbes. PCR-DGGE artifacts require optimization of the method when aiming at the phylogenetic identification of the selected DGGE bands. The aim of our study was to develop a procedure which will increase the reliability of the identification. Samples of rumen fluid were used for the optimization since they contain a complex microbial community that supports the generation of artifactual bands. An optimized procedure following band excision and elution of microbial DNA is proposed including nuclease treatment, selection of DNA polymerase with proofreading activity, and cloning prior to sequencing and identification analysis.
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