BioVisible offers a unique analysis service for the evaluation of the effects of probiotics and prebiotics on Gut microflora using FISH (fluorescence in situ hybridization), (T)DGGE ((Temperature)Denaturing Gradient Gel Electrophoresis) and/or Real-time PCR.
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Total DNA is isolated from fecal samples. Using universal primers a discriminating region of the 16S rDNA is amplified. The amplified product is applied on a T(D)GGE gel. After the gelelectrophoresis the gel is silver stained and ready for analysis.
The gel is scanned and a profile is generated based on band location and intensity.
The profiles are compared and a cluster analysis is performed.
The cluster analysis gives a measure of relatedness of the different fecal profiles. Especially within one subject a T(D)GGE cluster analysis can show the dynamics of the gut microbiota, in time, in relation to diet changes, Pro- Pre-biotica and/or drug administration.
Temperature Gradient Gel Electrophoresis (TGGE) or Denaturing Gradient Gel Electrophoresis (DGGE) is a molecular fingerprinting method that separates polymerase chain reaction (PCR)-generated DNA products. In TGGE there is a temperature gradient across the gel. in DGGE there is a chemical gradient across the gel. TGGE and DGGE are useful for analyzing nucleic acids such as DNA and RNA, and sometimes for proteins.
The polymerase chain reaction of environmental DNA of a single target can generate templates of differing DNA sequence that represent many of the dominant microbial organisms. However, since PCR products from a given reaction are of similar size (bp), conventional separation by agarose gel electrophoresis results only in a single DNA band that is largely non-descriptive. T(D)GGE can overcome this limitation by separating PCR products based on sequence differences that results in differential denaturing characteristics of the DNA.
During T(D)GGE, PCR products encounter increasingly higher temperature or concentrations of chemical denaturant as they migrate through a polyacrylamide gel. Upon reaching a threshold temperature or denaturant concentration, the weaker melting domains of the double-stranded PCR product will begin to denature at which time migration slows dramatically. Differing sequences of DNA (from different bacteria) will denature at different temperature or denaturant concentrations resulting in a pattern of bands. Each band theoretically representing a different bacterial population present in the community.