Winner of an R&D 100 Award in 2008, the PhyloChip identifies individual microbial species on the basis of the 16S rRNA gene, which is present in all microbes.
"Small regions of DNA base-pair sequence differences within the 16S rRNA gene can be used to distinguish different microbial species," Andersen says. "Because this gene encodes an integral structural component of the ribosome, it is tied directly to a microbe's metabolic activity."
In their first PCR-free analytical method, the Berkeley Lab researchers directly placed microbial rRNA on the PhyloChip. This offered the simplest and least biased view of the most metabolically active members of the microbial community in the sample.
"The rRNA can be thought of as the first responders to a changing set of environmental conditions that result in an increase or decrease in demand for new proteins," Andersen says. "Essentially, the greater the demand for new proteins, the greater the concentration of rRNA in the cell. This can be used as a measure of metabolic activity to show what microbes are responding the fastest to a new set of conditions. Slight changes in nucleotide sequences, analogous to changes in a bar code, are used to differentiate one organism from another."
One drawback to this method is that RNA binds much more tightly to the PhyloChip probes than is typical for DNA. This results in increased background noise and variability that can pose challenges. In response, the Berkeley Lab researchers developed a second PCR-free method in which the rRNA is replaced with a complementary DNA sequence (cDNA). The cDNA complement is then synthesized to create a double-stranded cDNA fragment that closely mirrors the properties of a PCR amplified 16S rRNA gene fragment.
"This approach has the advantages of decreased background noise and variability while maintaining a faithful representation of the naturally occurring, metabolically active microbial communitie
|Contact: Lynn Yarris|
DOE/Lawrence Berkeley National Laboratory