Anyone who has watched one of several CSI: Crime Scene Investigation television shows is aware that PCR (Polymerase Incidents) is usually a technology helpful to amplify the particular samples of DNA into forensic evidence that could identify perpetrators or victims of any crime. Microbiologists likewise use PCR to get the identity of microbes in samples extracted from a variety of sources for any lots of purposes. However, for microbial analysis, the use of PCR technology can cause problems. Now, researchers using the U.S. Us department of energy (DOE)’s Lawrence Berkeley National Laboratory (Berkeley Lab) have overcome those problems with the development of PCR-free technology that is certainly depending on Berkeley Lab’s award-winning, high-density DNA-based microarray the PhyloChip.
“We’ve developed two strategies to PCR-independent microbial community analysis with all the PhyloChip,” says Gary Andersen, a microbial ecologist with Berkeley Lab’s Earth Sciences Division. “Each method represents a simple and economical solution to directly query microbial communities in natural environments every affords the additional benefit from detecting probably the most metabolically active microbes in a community, the methods more than likely to attenuate toxins, drive biogeochemical cycles or proliferate in disease states.”
Andersen would be the corresponding author of your paper published inside journal Applied and Environmental Microbiology that discusses this research. The paper is titled “PCR amplification-independent means of detection of microbial communities because of the high density microarray PhyloChip.” Co-authoring the paper were Kristen DeAngelis, Cindy Wu, Harry Beller, Eoin Brodie, Romy Chakraborty, Todd DeSantis, Julian Fortney, Terry Hazen, Shariff Osman, Mary Singer and Lauren Tom.
How omnipresent are microbes from the environment? Extract a gram of soil from just about anywhere and you may anticipate finding many an incredible number of individual microbes representing greater thousand different taxa. On land or perhaps in water, from tropical forests to backyard compost heaps, these microbial communities will be the primary drivers of various ecosystem processes via their ability to decompose organic matter or catalyze important chemical reactions.
“With such complexity in the middle of a great number of ecosystem processes, there is a great must be in a position to reliably and affordably analyze microbial communities in samples from different environments,” Andersen says.
Scientists identify the average person microbial taxa in the sample via the genetic signatures of ribosomal RNA (rRNA), that is the genetic portion of the ribosome, the machinery in just a biological cell that makes protein array. PCR technology has typically been helpful to amplify the DNA within a sample to secure a sufficient level of genetic material for analysis.
“PCR amplification of microbial DNA introduces well-known distortions,” says Kristen DeAngelis, lead author of the AEM paper that’s with the University of Massachusetts but remains a collaborator with all the Joint BioEnergy Institute (JBEI), a DOE Bioenergy Research facility, which is led by Berkeley Lab. “As an example, DNA extracted from natural environments can sometimes include DNA from microbial populations which can be dead, dormant, or elsewhere indirectly triggering an ecosystem’s function.”
Andersen, DeAngelis and their colleagues about this project were able to remove the need for problematic PCR amplification using the PhyloChip. Put together by AEM paper co-authors Andersen, DeSantis and Brodie, plus Yvette Piceno, all with Berkeley Lab’s Earth Sciences Division, the PhyloChip is really a square-shaped microarray chip the size of one fourth. The most recent version is packed exceeding one million individual probes which enables it to be used to quickly, accurately and comprehensively detect the existence of approximately 50,000 different species of bacteria and archaea within a sample from any environmental source, without the need of culturing.
Winner of R&D 100 Award in 2008, the PhyloChip identifies individual microbial species judging by the 16S rRNA gene, which can be seen in all microbes.
“Small areas of DNA base-pair sequence differences inside the 16S rRNA gene may be used to distinguish different microbial species,” Andersen says. “As this gene encodes a vital structural component of the ribosome, it can be tied straight away to a microbe’s metabolic activity.”
Of their first PCR-free analytical method, the Berkeley Lab researchers directly placed microbial rRNA within the PhyloChip. This offered the simplest and least biased view of the most metabolically active members from the microbial community from the sample.
“The rRNA can be often considered as the first responders to some changing list of environmental conditions that bring about a boost or loss of need for new proteins,” Andersen says. “Essentially, the greater the demand for new proteins, greater the power of rRNA within the cell. This is often used as being a way of measuring metabolic activity showing what microbes are responding the fastest with a new pair of conditions. Slight modifications in nucleotide sequences, analogous to modifications to a Universal Product Code, are utilized to differentiate one organism from another.”
One drawback to this technique is always that RNA binds a lot more tightly for the PhyloChip probes than is normal for DNA. This results in increased ground noise and variability which could pose challenges. In reaction, the Berkeley Lab researchers created a second PCR-free method that the rRNA is replaced with a complementary DNA sequence (cDNA). The cDNA complement will be synthesized to create a double-stranded cDNA fragment that closely mirrors the properties of a PCR amplified 16S rRNA gene fragment.
“This method contains the benefits of decreased background noise and variability and a faithful representation from the natural, metabolically active microbial communities,” Andersen says. “The option of which these two PCR-free techniques to me is case-by-case. Some samples work fine while using the simple and easy and direct rRNA method although some require the excess steps in the double-stranded cDNA method.”
Andersen, DeAngelis and their colleagues successfully tested their two new PCR-free analysis techniques by comparing results with PCR-amplified DNA tests utilizing a mock community of eight microbial taxa in three environmental sample types: chromium-contaminated aquifer groundwater; tropical forest soil; and secondary sewage in sea water.
“Community profiles that is generated by our alternative methods showed taxa differences that could be expected dependant on accompanying data, but were missing in PCR-amplified communities,” DeAngelis says. “Our result show that a
PCR-independent view of microbial communities could possibly change which microbes the world thinks are dominant within an ecosystem. At JBEI, for example, we can easily start using thise new methods to identify microbes which can be most active in becoming worn feedstock biomass, such as switchgrass, into sugars which can be synthesized into advance biofuels.”