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Pyrosequencing is a DNA sequencing technique that utilizes enzyme coupled reactions and bioluminescence to monitor the pyrophosphate release accompanying nucleotide incorporation, in real time. The pyrosequencing method was developed by the group of Pal Nyren. With the aim of commercializing the new DNA sequencing technique, Nyren and others founded Pyrosequencing AB in 1997. The technology was licensed to 454 Life Sciences, which was later acquired by Roche in 2007. 16S DNA amplicon sequencing enables the identification and quantification of bacteria within complex mixtures of PCR amplicons. You can find a detailed description of the technique here .
Ion Torrent PGM sequencing:
The Ion Torrent sequencing platform from Life Technologies employs First PostLight™ sequencing technology (semiconductor technology). The detection of a specific nucleotide on a growing DNA strand occurs inside a well of an Ion Torrent semiconductor chip. The incorporation of the correct base triggers a chemical reaction that releases a hydrogen ion as byproduct, which in turn changes the pH of the solution. The Ion sensor captures voltage change measurements from the direct release of hydrogen ions following DNA polymerization and incorporation events get recorded as digital signals 1 and 0. This technology differs from other sequencing technologies in that no modified nucleotides, no lasers, no optics, no camera, no fluorescence, no enzyme cascade are used.
The method is based on the conserved 16S rDNA gene present in all bacteria (a comprehensive list of 16S rDNA sequences from gut bacteria have been deposited and categorized in the Ribosomal Database Project II ). By calculating the number of conserved 16S rDNA genes, qPCR can indirectly indicate the number of bacteria present in a given sample. Additionally, appropriate design of qPCR oligonucleotide probes (primers) that can identify the sequence of the 16S gene variable regions allows for the amplification of genes from a specific bacterium of interest whilst excluding all other bacteria, making qPCR a useful tool for quantifying specific bacteria in a complex microbial community.
In order to confirm bacterial identity, PCR-amplified 16S rDNA fragments can be cloned into plasmids and transformed into an appropriate host (usually E. coli). Each transformed E. coli colony will contain only one 16S rDNA PCR-originated fragment from the original sample. A number of colonies are picked randomly and the insert is sequenced. The DNA sequence is then compared to a database (for example RDP II) for identification by DNA homology.
Terminal Restriction Fragment Length Polymorphism (TRFLP) Analysis
TRFLP is a method that allows the identification of major phylogenetic groups in complex microbial communities. It is based on the treatment with restriction enzymes of a DNA fragment specifically amplified by PCR. The terminal fluorescent-labeled fragments produced are separated by size using a DNA sequencer. Each specific bacterial group will produce a specific profile that can be identified by comparing them to existing profile databases.
Consulting and Research Support
- Sample genomic DNA isolation
- Plasmid and virus nucleic acids isolation
- RNA isolation
- Strain typing
- Optimization of bacterial culture conditions
- High-throughput liquid handling (PCR reaction set up, sample pooling, picogreen quantification of nucleic acids)