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.
Please contact us for more information
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.
The 16S ribosomal RNA genes of most bacteria are by approximation equal in size but exhibit distinct variation in base-pair composition. The ribosomal RNA genes of bacteria therefore represent unique targets for identifying species-specific sequences. Besides large variations very conserved sequence regions are also discernible. In fact, the ribosomal RNA’s represent a mosaic of highly variable and conserved regions. This feature can be used to separate the ribosomal RNA genes present in a mixed population of bacteria on the basis of DGGE.
In essence, DGGE is based on the principle that, in a polyacrylamide matrix, double stranded DNA’s migrate at a higher rate than their (partially) single stranded counterparts. The introduction of a G+C-rich “clamp” to one side of the double stranded DNA will keep the two strands connected to each other even when relatively strong denaturing conditions are met. Due to this GC-clamp, the difference in migration rate between double stranded and single stranded DNA’s is dramatically improved. In fully denatured form, the migration of the DNA fragments is virtually arrested.
The possibility to effectively separate many (>20) different ribosomal RNA genes is further based on the principle that strand separation or denaturation of the dsDNA’s is influenced by the G+C content of the DNA’s and the presence of “melting regions” (i.e., partially A+T-rich sequences at which strand separating is initiated earlier than in the rest of the helix thus retarding the migration of the fragment).
In order to obtain the (partial) ribosomal RNA genes from all the bacteria in a complex mixed population, the genomic DNA extracted from the total population is used as a template in a PCR reaction. Rigorous DNA extraction procedures should be used in order to ensure that genomic DNA is liberated from all bacteria. Specific regions of this mixed rRNA gene pool are amplified by using a PCR primer pair directed towards (universally) conserved gene sequences. Only one of the PCR primers contains the GC-clamp. After gel-electrophoretic separation of these PCR products on a chemical gradient of urea and silver staining of the DNA, a banding pattern emerges that represents the diversity of the ribosomal RNA gene sequences present in the sample. The intensity of the individual bands is a measure for the relative abundance of this sequence in the population. Every band originates - in principle - from one bacterial species, although certain PCR bias towards A+T-rich sequences may occur.
Computer analysis of the DGGE profiles of the samples results in similarity percentages between the individual samples. DGGE is a reproducible form of TGGE, in which the separation of DNA’s is not dependent on a temperature gradient but on a chemical gradient of urea.
DGGE is an exceptional tool to study the bacterial species composition of unknown samples. By inter-sample comparison, dominant shifts in population composition can be monitored without any prior knowledge of that sample. By using DGGE, bacterial population dynamics can be studied in more detail.
BioVisible starts a new analysis service using the Mitox™ DNA - Mitochondrial toxicity assay to measure simultaneously the nuclear DNA and mitochondrial RNA content of the cell. This assay can be used as a biomarker for toxicity and/or efficacy of antiviral treatment.
This analysis service is offered in collaboration with Primagen. Click Logo for preliminary website.
Did you know; The human brain stops growing at the age of 18